Mutated class I major histocompatibility proteins and complexes

ABSTRACT

Provided herein are combinatorial libraries containing chimeric Major Histocompatibility Comples (MHC) Class I proteins displayed on the surfaces of recombinant yeast cells. Members of the libraries, especially where those libraries have been mutagenized either with error-prone Polymerase Chain Reaction or with site-directed oligonucleotide mutagenesis, are improved in conformation stability or in binding to a target, e.g., a peptide or other ligand as compared with the stability or binding affinity of a wild type MHC Class I chimeric protein. The improved mutant chimeric proteins can be selected by various means, including fluorescence activated cell sorting with a fluorescent ligand bound to the surfaces of the yeast cells displaying the improved mutant chimeric protein.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation-in-Part of U.S. applicationNo. 60/254,495, filed Dec. 8, 2000.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

[0002] This invention was made, at least in part, with funding from theNational Institutes of Health (Grant No. PHS 5 RO1 AI35990).Accordingly, the United States Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0003] The field of the present invention is molecular biology, inparticular, as it is related to combinatorial libraries of immune cellproteins displayed on the cell surface of a recombinant host cell. Morespecifically, the present invention relates to a library of majorhistocompatibility locus proteins displayed on the surfaces ofrecombinant yeast cells, to mutant MHC Class I and proteins selected forimproved binding to particular target peptides, to mutant MHC Class Iproteins selected for binding to a particular antigen, to MHC Class Iproteins of improved stability and to the use of the selected highaffinity and/or more stable MHC derivatives in diagnostic methods andimaging assays, among other applications including prophylactic andtherapeutic treatments.

[0004] Proteins encoded by the major histocompatibility complex (calledMHC proteins) are requisite components of the antigenic complexes thatare involved in many diseases. These diseases include cases where thebody reacts with one's own MHC proteins (in various autoimmune diseases)or infectious diseases and cancer, where the MHC are critical in bindingand presenting foreign, antigenic peptides. In this invention, MHCproteins of the class I type were expressed as heterologous,surface-linked fusions on yeast cells with the goal of generatingimproved MHC proteins. Libraries of mutant MHC and mutant peptide-MHCcomplexes could be screened for higher surface levels in order toidentify variants that exhibited improved properties, including enhancedstability. For the first time, this system allows the directed evolutionof MHC molecules that represent novel agents for various diagnostic andtherapeutic applications. These agents could be used in cancer,infectious diseases (e.g., virus infections), and autoimmune diseases(e.g., multiple sclerosis, type I diabetes, rheumatoid arthritis).

[0005] T cell receptors (TCRs) and antibodies have evolved to recognizedifferent classes of ligands. Antibodies function as membrane-bound andsoluble proteins that bind to soluble antigens, whereas in nature, TCRsfunction only as membrane-bound molecules that bind to cell-associatedpeptide/MHC antigens. All of the energy of the antibody:antigeninteraction focuses on the foreign antigen, whereas a substantialfraction of the energy of the TCR:peptide/MHC interaction seems to bedirected at the self-MHC molecule [Manning et al. (1998) Immunity8:413:425]. In addition, antibodies can have ligand-binding affinitiesthat are orders of magnitude higher than those of TCRs, largely becauseof the processes of somatic mutation and affinity maturation. In theirnormal cellular context, TCRs do not undergo somatic mutation, and theprocesses of thymic selection seem to operate by maintaining a narrowwindow of affinities [Alam et al. (1996) Nature 381:616-620]. Theassociation of TCRs at the cell surface with the accessory molecules CD4or CD8 also may influence the functional affinity of TCRs [Garcia et al.(1996) Nature 384:577-581]. Despite these differences, thethree-dimensional structures of the two proteins are remarkably similar,with the hypervariable regions forming loops on a single face of themolecule that contacts the antigen.

[0006] There is a long felt need in the art for Class I MHC proteins andClass I MHC/peptide complexes with improved stability and/or withimproved T cell regulatory properties. Such improved Class I MHCproteins or complexes are useful in activating T cells that participatein the removal of target cells including neoplastic cells and cellsinfected with pathogenic agents including, but not limited to, viruses,protozoans, bacteria, fungi or nematodes. The improved Class I MHCproteins and complexes of the present invention are also improved foruse as research tools.

SUMMARY OF THE INVENTION

[0007] The present invention provides combinatorial libraries of Class IMHC proteins displayed on the surfaces of recombinant host cells, forexample, yeast cells, desirably, Saccharomyces cerevisiae. From such alibrary can be isolated mutant MHC proteins that exhibit a relativelyhigh affinity for a peptide ligand of interest. Also within the scope ofthe present invention are methods for isolating mutant Class I MHCproteins with improved stability, especially as MHC/peptide complexes ofimproved stability.

[0008] The present invention further provides Class I MHC/peptidecomplexes and proteins that exhibit increased stability over the wildtype Class I MHC/peptide complex or MHC protein, and which MHC proteinsexhibit high affinity for a peptide ligand interest. This ligand can bea peptide, a protein, a carbohydrate moiety, or a lipid moiety, amongothers.

[0009] Suitable labels allowing for detection of a ligand bound to anMHC protein, directly or indirectly, include but are not limited tofluorescent compounds, chemiluminescent compounds, radioisotopes,chromophores, and others. The stable, MHC protein of the presentinvention, where it specifically binds to a tumor cell antigen with highaffinity and specificity can be used in diagnostic tests to detect Tcells that are specific for the Class I MHC/peptide complex. The Class IMHC/peptide complexes of the present invention can also be used toactivate T cells and thus, to enhance an immune response to an antigenor target cell of interest.

[0010] Also provided by the invention are novel yeast display vectorsand surface expression constructs in which the portion of the fusionprotein that mediates attachment to the cell surface (the AGA2 sequence)is located downstream, or at the carboxy end of the protein/sequence ofinterest. See FIG. 2.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 provides a ribbon diagram corresponding to the crystalstructure of a class I major histocompatibility complex with highlightedsubunits. The MHC is trimeric. The MHC K^(b) α chain (^(˜)350 aminoacids) binds to an 8-10 amino acid residue peptide in the peptidebinding cleft. The β2 microglobulin (99 amino acids) associatesnon-covalently with the α chain.

[0012]FIG. 2 illustrates the displayed MHC protein of interest displayedon the yeast cell surface via a disulfide linkage through the AGA2portion of the fusion protein comprising the MHC component.

[0013]FIG. 3 illustrates diagrammatically the pCT302 yeast surfacedisplay vector which contains a sequence encoding AGA2/HA-Class IMHC-c-myc fusion protein. This fusion protein coding sequence isexpressed in yeast under the regulatory control of the GAL1-10 promoter.A similar vector, pYD1, is commercially available from Invitrogen.

[0014]FIG. 4 provides diagrams of various single-chain Class I MHCconstructs cloned into the pCT302 Yeast Display Vector.

[0015]FIG. 5 provides the results of flow cytometric analyses of variousK^(b) constructions. Yeast cells displaying K^(b)/β2m, SIYR K^(b)/β2m,dEV8 K^(b)/β2m, and OVA K^(b)/β2m were stained with anti-c-myc Mab 9E10and biotinylated anti-K^(b) antibody (B.8.24.3) followed by FITC labeledF(ab′)₂ goat anti-mouse IgG or SA-PE (shaded peaks). For a backgroundcontrol, yeast were treated with only the secondary stain (unshadedpeak). Labeled yeast were analyzed on Coulter Epics XL flow cytometer.All four constructions displayed the properly folded K^(b) molecule onthe surface.

[0016] FIGS. 6A-6D illustrate histograms from CD69 up-regulation assays.Induction of T cell activation marker CD69 on naive splenocytes (10⁶) inthe presence of K^(b)/β2m, anti-TCR scFv, and SIYR/K^(b)/β2m yeastcells. Induced yeast cells (10⁶) were incubated at 37° C. and 5% CO₂with splenocytes from 2C TCR/RAG-1^(−/−) mice. After 20 hours inculture, cells were harvested, washed in PBS/0.5% BSA and stained withbiotinylated clonotypic antibody, 1B2 [Kranz et al. (1984) Proc. Natl.Acad. Sci. USA 81: 7922-7926] for 45 minutes. These yeast were washedand stained with a mixture of the early activation marker FITC anti-CD69antibody and SA:PE. The yeast/T cell mixture was analyzed for boundFITC-labeled anti-CD69 antibody by flow cytometry, gating on 1B2positive T cells. The mean fluorescence units for FITC anti-CD69antibody in the absence of yeast, presence of K^(b)/b2m yeast, anti-TCRscFv yeast, and SIYR/K^(b/)β2m yeast are indicated and illustrated inthe histograms. Yeast cells that expressed K^(b) with the agonistpeptide (called “SIYR”; SIYRYYGL SEQ ID NO:1), but not yeast cells withK^(b) and no peptide, were capable up-regulating the CD69 molecule onthe 2C T cells. This up-regulation was even greater than observed withthe positive control anti-TCR antibody KJ16 [Cho et al. (1998) J.Immunol. Methods 220(1-2): 179-188].

[0017]FIG. 7 shows the results of direct activation of T cells by yeastthat express Class I Peptide/MHC, as measured by flow cytometry.Up-regulation on 2C TCR/RAG^(−/−) splenocytes incubated with varyingratios of yeast (3 to 100×10⁵ yeast and 106 T cells) were analyzed byflow cytometry. The mean fluorescence units of FITC anti-CD69 antibodywas detected on 1B2 gated T cells following 20 hours of incubation (37°C., 5% CO₂) of 2C TCR/RAG^(−/−) splenocytes with yeast that expressed:SIYR/K^(b)/β2m, anti-TCR scFv (positive control), and K^(b)/β2m(negative control).

[0018]FIG. 8 shows direct activation of T cells by pMHC on yeast, thusproviding evidence for pMHC specificity excess peptide and anti-K^(b)antibody inhibit CD69 up-regulation. In order to confirm the specificityof T cell activation, K^(b)/β2m (negative control), SIYR/K^(b)/β2mbearing yeast cells, and 2C TCR/RAG^(−/−) splenocytes were incubatedwith excess OVA (SIINFEKL, SEQ ID NO:2) peptide or anti-K^(b) antibody(50 μg/ml B.8.24.3) at 37° C., 5%CO₂. Excess OVA peptide binds to K^(b)and should compete for binding to the K^(b) molecule, but it is notrecognized by 2C T cells. Anti-K^(b) antibody recognizes the al/a2helices and should prevent binding by the T cell receptor from 2C Tcells. After 1 hour, 2C TCR/RAG^(−/−) splenocytes were mixed with theyeast and incubated for 20 hrs. Detection of CD69 on 2C T cellsfollowing incubation with yeast in the absence or presence of inhibitorswas detected with FITC anti-CD69 antibody and reported as the meanfluorescence units. Both excess OVA peptide and the anti-K^(b) antibodyinhibited the up-regulation of CD69, confirming that the recognition ofthe yeast-bound SIYR/K^(b) was specific.

[0019]FIG. 9 diagrammatically illustrates cloning and transformation viahomologous recombination. Linear mutagenized MHC protein coding sequenceand linearized pCT302 vector are co-electroporated into yeast. Thisstrategy is used in the preparation of the yeast display library for theMHC Class I protein.

[0020]FIG. 10 illustrates the strategy for mutagenizing a yeast displaylibrary and screening that library by taking advantage of the bindingproperties of mutant vs wild type displayed proteins. Fluorescentlylabeled ligands or antibodies which bind the displayed protein areemployed in flow cytometry assays.

[0021]FIG. 11 provides examples of sorting libraries generated by randommutagenesis. A SIYR/K^(b)/β2m error prone library (SEP) and adEV8/K^(b)/β2m error prone library (dEP) were each sorted 3 times. TheSEP and dEP yeast (10⁷), after having been induced for 2 days, werewashed with PBS/0.5% BSA, stained with biotinylated anti-K^(b)(B.8.24.3) for 60 min, washed again, and stained with SA:PE for 30 min.The first sort isolated the top 1.0% of the population using a 1:25dilution of anti-K^(b). The second and third sorts were stained with a1:250 and 1:500 dilution of anti-K^(b) for SEP and 1:250 dilution forboth the second and third sort of dEP. The second and third sortsisolated the top 0.25% and 0. 1% of the population, respectively.Representative histograms from the second and third sort of the SEPlibrary illustrate the mean fluorescence shift of the total populationor enhancement of the more fluorescent yeast cells from sort 2 to sort3. This indicated that there were likely to be K^(b) mutants thatexhibited increased stability and hence, increased surface levels[Shusta, (1999) J. Mol. Biol. 292: 949-956].

[0022]FIG. 12 shows the K^(b) surface levels of mutant clones isolatedby sorting from dEV8/K^(b) error prone PCR library. Following sorting,ten randomly selected clones from the dEV8/K^(b/)β2m library wereanalyzed on the flow cytometer for binding to the biotinylatedanti-K^(b) antibody (B.8.24.3). Binding was detected with SA:PE, andmean fluorescence for each clone was determined.

[0023]FIG. 13 shows sequences of mutant clones isolated by sorting fromdEV8/K^(b) error prone PCR library. The mutations in three clonesselected from the dEV8/K^(b)/β2m error prone library (sort 3) and thecrystal structure with two of the mutations (W167R,Y63N) are shown.

[0024]FIG. 14 illustrates construction of a directed, mutagenic peptidelibrary in dEV8/K^(b) library construction. Mutagenic PCR of positions1-3 of the peptide dEV8 was performed using the degenerate upstreamprimer (5′ ATA TTT TCT GTT ATT GCT TCA GTT TTA GCA GCT AGC TTG GAT AAAAGA NNS NNS NNS AAA TTC 3′, SEQ ID NO:3) and a downstreamvector-specific primer. Using homologous recombination, the mutagenicdEV8/K^(b)/β2m PCR product and Nhe I-Nde I digested dEV8/K^(b)/β2mpCT302 were electroporated into electrocompetent yeast (BY5465, strainEBY100). A dilution of the transformed library was plated on SD-CAAplates and incubated for 3 days at 30° C. to obtain the library size(7×10³).

[0025]FIG. 15 shows the properly folded α3 domain, but the α1/α2 domainsof L^(d) are not displayed on yeast. L^(d)/β2m yeast cells were pulsedfor two hours with the L^(d)-binding peptide QL9 (QLSPFPFDL, SEQ IDNO:4), washed with PBS/0.5%BSA, and stained with anti-L^(d) antibodies,28.14.8 (α3 specific) or 30-5-7 (α1/α2 specific). After 45 min, cellswere washed and stained with FITC-labeled F(ab=)₂ goat anti-mouse IgG,and binding was detected using flow cytometry. The histograms generatedfrom the L^(d)/β2m displaying yeast represent the binding of the α3specific antibody (28.14.8). The 30-5-7 antibody, α1/α2 specific, didnot show the same binding (histograms not included), suggesting that atleast one of these two domains was not folded properly and thus might bestabilized by a process of directed evolution and yeast display.

[0026]FIG. 16 illustrates the result of sorting a yeast display mutantlibrary of “unstable” Class I MHC L^(d). Yeast cells displayingL^(d)/β2m were induced at 20 C for 2 days in the presence of anL^(d)/β2m specific peptide, QLSPFPFDL (QL9, SEQ ID NO:4). L^(d)/β2myeast were stained with supernatants from D. melanogaster cellsexpressing the high-affinity T cell receptor (m6), biotinylated anti-Tcell receptor antibody (F23.1) and a streptavidin-phycoerythrin (SA:PE)conjugate (shaded peak). The L^(d)/β2m yeast cells were also stainedwith an α2 domain specific antibody (30-5-7) and an α3 domain specificantibody (28.14.8) followed by a FITC labeled F(ab′)₂ goat anti-mouseIgG (shaded peak). For a background control, yeast cells were treatedwith only the secondary stains (unshaded peaks). Labeled yeast cellswere detected using a Coulter EPICS XL flow cytometer. The L^(d)/β2m α3domain was folded properly on the surface, but not the α2 domain or theTCR binding domain.

[0027]FIG. 17 demonstrates that the L^(d) mutant 30.8 exhibits aproperly folded TCR binding domain and properly folded α2/α3 domains. AnL^(d)/β2m error prone library was created using homologous recombinationand electroporation into electrocompetent S. cerevisiae EBY100 cells.The L^(d)/β2m error prone yeast library was sorted with L^(d) /βb 2m α3specific antibody, 28.14.8, and an L^(d)/β2m α2 specific antibody,30-5-7. Sorted yeast cells were screened by flow cytometry for bindingto 30-5-7 antibody and high affinity T cell receptor supernatant (m6),as described in FIG. 16 above. Unlike yeast cells that express the wildtype L^(d)/β2m, the L^(d)β2m mutant, yeast cells expressing 30.8 weredetected by the α2 specific antibody (30-5-7) and by the high affinity Tcell receptor (m6) when the correct peptide (QL9) was added exogenously.Mutant 30.8 was also detected by the α3 specific antibody, 28.14.8.

[0028]FIG. 18 summarizes the results for a yeast display library ofpeptides that bind to MHC class I K^(b). Known K^(b) peptides (SIYRYYGL,SEQ ID NO:1; EQYKFYSV, SEQ ID NO:5; SIINFEKL, SEQ ID NO:2) have anchorresidues at peptide positions 5 and 8. P5 requires an aromatic aminoacid whereas P8 requires a hydrophobic amino acid. Yeast displaying aK^(b) peptide binding motif, but with the AGA2 fused at the aminoterminus, were isolated after incubation of yeast cells with the dEV8(EQYKFYSV)/K^(b) tetramer.

[0029]FIG. 19 shows that yeast cells displaying the K^(b) peptide motifwere detected by flow cytometry using a fluorescent-labeled K^(b)tetramer. Yeast cells with the C-terminal sequence shown in the toppanel were stained with dEV8 (EQYKFYSV)/K^(b) tetramers for 12 hours.The dEV8 peptide dissociates from the K^(b) pocket, and the AGA2-fusedpeptide on the yeast surface binds to the free K^(b). Binding of thepeptide motif to the K^(b) tetramer was detected by flow cytometry(shaded peak). For a background control, yeast cells were treated withstreptavidin-phycoerythrin (SA:PE) conjugate alone. Mutagenicsubstitution of the proline residue at position P4 with an alanineresidue reduced binding significantly (bottom panel), indicating thatthe prolione is important in K^(b)-binding.

[0030]FIG. 20 shows that novel class I MHC binding peptides are isolatedby yeast display technology. Using the lead peptides identified by yeastdisplay experiments (FIG. 19), one can now identify improved peptides byproduction of libraries that contain mutations in non-anchor residues(e.g. P6, P7, and P9).

DETAILED DESCRIPTION OF THE INVENTION

[0031] The role of proteins encoded by the major histocompatibilitycomplex (called MHC proteins) has now been known for almost twentyyears. MHC proteins are expressed by every individual and function as“antigen-presenting” molecules. That is, each MHC protein can bind to avariety of different small peptides (8 to 20 amino acids in length) thatare derived from proteins present inside a cell. MHC proteins presentboth self-peptides (i.e., derived from an individual's own endogenousproteins) and foreign peptides (i.e., derived from a foreign agent suchas a virus). Once a peptide is bound to the MHC protein, the entirepeptide-MHC complex is expressed on the surface of the cell. If thepeptide is foreign, a T lymphocyte (T cell) can potentially recognizethe complex, and the resultant interaction of the T cell receptor (TCR)and the pMHC can result in T cell activation. T cell activation can leadto recruitment of other immune cells and a corresponding inflammatoryreaction. Such inflammatory reactions are beneficial if the pMHC targetantigen is, in fact, derived from an infectious agent or from atransformed cell (i.e., cancer). However, such inflammatory reactionscan be very detrimental if the pMHC target antigen is derived from selftissue, as the reactions can lead to severe autoimmune disease, where anindividual's immune system attacks normal tissue. Such is the case whena patient's lymphocytes attack the islet cells of the pancreas (type Idiabetes), the nervous system (multiple sclerosis), or joint-derivedcomponents (rheumatoid arthritis).

[0032] The central role of pMHC complexes in these phenomena has beenestablished by thousands of published studies that include geneticlinkages of diseases to the human MHC locus (HLA). It has now also beenestablished that it is possible to use appropriately characterizedpeptide-MHC molecules as either agonists of an immune response (e.g., incancer and infectious diseases) or as antagonists of responses (e.g., inautoimmune responses). While several approaches have been taken toproduce such pMHC complexes in soluble forms for these purposes and forbiochemical/structural studies, it has not been possible to use currentmethods of in vitro directed evolution to improve the stability orantigenicity of the pMHC complex. This is because the pMHC complex isnormally a membrane-associated complex composed of multiple differentsubunits (heavy chain, beta-2-microglobulin, and peptide in the case ofa class I MHC and α-chain, β-chain, and peptide in the case of class IIMHC) and such proteins are typically not amenable to the current methodsof directed evolution (primarily phage display). The present inventionshows that a display system for directed evolution can be used toexpress properly folded Class I MHC proteins on the surface of yeast.The displayed peptide-MHC complexes can be used to directly activate Tcells, in order to identify/screen for pMHC antigens. In addition,mutated libraries of the pMHC proteins could be created and used forselection by flow sorting of stabilized pMHC variants. The stabilizedvariants can be identified because they were expressed at higher levelson the yeast surface and can therefore be easily identified by using afluorescent-labeled probe for the pMHC construct, combined withhigh-throughput flow cytometric sorting or such cells.

[0033] The Class I MHC proteins (see FIG. 1 for ribbon diagram) arecomposed of an α chain of 350 amino acids and a β2m chain of 99 aminoacids; the two chains are noncovalently associated with one another.Peptides that are about 8-10 residues bind the Class I MHC molecules.About 10⁵ to 10⁶ peptide-Class I MHC complexes are displayed on thesurfaces of nucleated cells. Class I MHC complexes are involved in therecognition of virus infected cells, pathogen-infected cells, and tumorantigens by cytotoxic T lymphocytes.

[0034] To date, no Class I MHC protein has been crystallized without apeptide bound in the peptide binding cleft. Some Class I MHC proteinsare difficult or impossible to produce in soluble form because of theirinstabilities. For example, the mouse Class I MHC K^(b) is relativelystable, and it has been used in many studies. By contrast, the mouseClass I MHC L^(d) is less stable, and it has been more difficult toproduce. The stability of a particular peptide-Class I MHC complex isdirectly related to its ability to stimulate efficient T cell responses,for example, in vaccine applications. A system for the in vitroevolution of more stable peptide-MHC complexes allows for novel agentsand vaccines to be used in stimulating protective immune responsesagainst diseases and neoplastic conditions.

[0035] The present invention allows the creation and isolation ofstabilized variants of peptide-MHC complexes. Toward this end, we havedisplayed single-chain peptide/Class I MHC (α chain/β2m) complexes onthe surface of yeast cells. Yeast cells expressing a specificpeptide/MHC complex (SIYR/K^(b)) on the cell surface were capable ofdirectly activating T cells, thus providing a method for screeningpowerful T cell agonists. Mutant libraries of a less stable peptide/MHCcomplex (dEV8/K^(b)) were made and expressed, and mutant MHCpolypeptides of increased stability were isolated. Similarly, the lessstable MHC L^(d) was expressed as a mutant library displayed on thesurface of recombinant yeast cells, and more stable L^(d) variants wereisolated using flow cytometry screening methodology. WO 99/36569,incorporated by reference herein, provides abundant discussion of thisdisplay technology.

[0036]FIG. 2 illustrates the MHC protein of interest displayed on theyeast cell surface via a disulfide linkage through the AGA2 portion ofthe fusion protein comprising the MHC component. AGA2 is a matingadhesion receptor which is naturally bound to the cell surface indisulfide linkage to the AGAL protein. The HA and the c-myc portions ofthe displayed fusion protein serve as epitope tags and can be used innormalizing the fluorescent peptide binding data. Each recombinant yeastcell displays about 50,000 copies of the surface-bound fusion protein(if stable) on its surface. A fluorescent antibody or peptide ligand isadded, and the cells are sorted using flow cytometry. Those MHC fusionproteins of increased stability exhibit stronger binding of thefluorescent ligand, and these cells are selected during the cell sortingprocedure.

[0037]FIG. 3 illustrates diagrammatically the pCT302 yeast surfacedisplay vector which contains a sequence encoding AGA2/HA-Class IMHC-c-myc fusion protein. This fusion protein coding sequence isexpressed in yeast under the regulatory control of the GAL1-10 promoter.A similar vector, pYD 1, is commercially available from Invitrogen(Carlsbad, Calif.).

[0038]FIG. 4 shows various constructs for the expression of fusionproteins containing portions representing the peptide binding portionsof various Class I MHC proteins. Surprisingly, the AGA2 portion wasfunctional either at the N-terminus or the C-terminus of the fusionprotein, and it mediated binding to the yeast cell wall surface whenassociated with any of the protein portions tested and in both theamino- or carboxyl terminal positions. In FIG. 4 SS refers to a signalpeptide sequence necessary for proper intracellular transport of thefusion protein. The signal peptide is cleaved prior to display on thecell surface. The MHC polypeptide coding portion includes a sequenceencoding a 15 amino acid spacer between the COOH terminus of the α chainand the NH₂ end of the P2m portion. HA refers to the peptide tag derivedin sequence from hemagglutinin, which tag is located at the aminoterminus of the a chain. C-myc refers to the peptide tag at the COOHterminus of the β2m portion. The three peptides linked at the aminoterminus of K^(b) represent a strong agonist (“SIYR” peptide, SEQ IDNO:1), a weak agonist (dEV8) and a null peptide (OVA) for the T cellclone called 2C. The AGA2 coding sequence was cloned at the COOH terminiof these constructs in order to allow free NH₂ termini of the peptides,which is generally thought to be important for proper binding to Class IMHC polypeptides. All fusion protein coding sequences were assembledusing standard polymerase chain reaction (PCR) strategies.

[0039] The correct foldings of various fusion display proteins wereconfirmed by fluorescence activated cell sorting after binding of therecombinant yeast cells to fluorescently labeled antibody specific forc-myc or K^(b). See FIG. 5.

[0040] To demonstrate the direct activation of T cells by recombinantyeast cells expressing a peptide/MHC complex, naive splenic T cells froma TCR transgenic mouse (2C TCR tg) were incubated with yeast cellsexpressing SIYR/K^(b), K^(b) (negative control) or anti-TCR scFv(positive control). After about 20 hours in culture, the T cells wereanalyzed by flow cytometry for the up-regulation of the activationmarker CD69. See FIGS. 6A-6D for a graphical display of the results.

[0041]FIG. 7 shows the dependence of T cell activation on T cells and onthe presence of a peptide/MHC complex. FIG. 8 demonstrates the pMHCspecificity: excess free peptide and anti-K^(b) antibody inhibit CD69up-regulation in T cells.

[0042] We have demonstrated direct activation of T cells by yeast cellsthat express peptide/MHC. Splenocytes (10⁶) from 2C TCR/RAG-1^(−/−) micewere combined with approximately 3-100×10⁵ of KF washed yeast cellsdisplaying K^(b)/β2m, SIYR/K^(b)/β2m, or anti-TCR scFv (positivecontrol) at 37 C, 5% CO₂ in a 24 well plate. After 20 hours in culture,cells were harvested, washed in PBS/0.5%BSA and stained withbiotinylated clonotypic antibody, 1B2 [Kranz et al. (1984) supra] for 45minutes. These yeast/T cell mixtures were washed and stained with amixture of the early activation marker FITC-labeled anti-CD69 antibodyand SA:PE. The yeast/T cell mixture was analyzed for the CD69 marker(present only on activated T cells) by flow cytometry and gating on 1B2positive T cells.

[0043] The yeast display system was exploited to produce a randommutagenized library from which stabilized mutant Class I MHC K^(b)sequences were isolated. Constructs encoding the fusion proteinscontaining SIYR/K^(b) and dEV8/K^(b) portions were mutagenized randomlyusing error prone PCR (0.16 Mn:Mg molar ratio). The homologousrecombination scheme illustrated in FIG. 9 was employed to create thelibraries. From the SIYR/K^(b) experiment, 3.6 million transformantswere recovered. From the dEV8/K^(b) experiment, 2.7 milliontransformants were recovered. 5 plasmid inserts from each mutatedlibrary were sequenced (about 3200 bp of sequence per library) todetermine the mutation frequency. In the SIYR/K^(b) experiment themutation frequency was 0.37% (2 wild type sequences, 3, 4, and 5 mutantsequences). In the dEV8/K^(b) experiment, there was a 0.06% mutationfrequency (4 wild type, 1 mutant sequence).

[0044]FIG. 10 diagrammatically presents the generic screening strategyfor screening the mutated yeast display libraries. FIG. 11 illustratesthe results for second and third sorts with anti-K^(b) monoclonalantibody B.8.24.3. In the second sort the top 0.25% of the cells(according to fluorescence intensity, mfu, mean fluorescence units) wereselected, and in the third sort, the top 0.1% of the cells wereselected. The profiles were similar for the dEV8/K^(b) experiment.

[0045]FIG. 12 illustrates different K^(b) binding as reflected indifferent levels of fluorescent peptide bound after sorting of thedEV8/K^(b) mutant library produced using error prone PCR. FIG. 13presents selected sequences of mutant clones isolated by sorting of thedEV8/K^(b) mutant library, and the positions of the mutations are shownon the ribbon diagram of the Class I MHC protein. The following providesfurther details on the construction of a dEV8/K^(b) mutant library. Fromthe ribbon diagram it is deduced that the sequence EQYKFYSV (SEQ IDNO:5) is important. The E (P1, the first amino acid of the peptidesequence) is buried in the first pocket. Q, P2, is directed down intothe pocket. Y, P3, is a bulky, secondary anchor residue. K, P3, is aprimary TCR contact, and it is directed out of the pocket. The P residue(P4) is an aromatic residue directed into the pocket; it serves as aprimary MHC anchor. The Y residue at P6 is a large aromatic residuewhich also functions in TCR contact. The S residue (P7) is a smallresidue; the small size is dictated by the space available. The last Vresidue (P8) serves as a primary anchor residue, and it is directed intothe pocket. To select mutations that stabilize dEV8 binding to K^(b),mutations are introduced in the library at positions that point intoK^(b). A degenerate library is produced at P1-P3 (i.e., NNNKFYSV, SEQ IDNO:6). That library is constructed by error prone PCR and the mutantlibrary is introduced into the wild type dEV8/K^(b) plasmid byhomologous transformation as shown in FIG. 9. Isolated recombinants arethen sequenced. Of four sequenced, each contained different nucleotidesencoding P1-P3. See FIG. 14 and its description herein above.

[0046] The motivations for using directed evolution to isolatestabilized variants of L^(d) include the lower stability of the wildtype L^(d) protein based on biochemical studies, the suboptimal loadingof peptides in the endoplasmic reticulum, the slower intracellulartransport of L^(d) (4 hr v 1 hr), the lower cell surface expression (2-4fold lower) and fewer α/β2m contacts. See Table 1.

[0047]FIG. 15 provides data demonstrating that the properly folded α3domain but not the α1/α2 domains of L^(d) are displayed on the surfaceof recombinant yeast cells. We have also demonstrated that anti-L^(d) α½domain (monoclonal antibody 30.5.7) did not bind to the same L^(d)/β2mdisplayed on yeast cell surfaces.

[0048] In summary, the Class I MHC K^(b) was cloned as an AGA2 fusion insingle chain format (AGA2-HA-K^(b)-β2m-c-myc). The full protein wasdetected on the yeast cell surface using an antibody that recognizes theproperly folded α1/α2 domains of K^(b). The coding sequences of thisprotein were also cloned with AGA2 at the carboxy terminus, with K^(b)binding peptides (SIYR, dEV8 and OVA) linked at the amino termini(peptide-HA-K^(b)-β2m-c-myc-AGA2). Yeast that expressed the T cellspecific peptide K^(b) on the cell surface were capable of directlyactivating T cells, thereby providing a system for screening T cellagonists.

[0049] To create stabilized K^(b) mutant fusion protein, two differentmutational libraries were produced by error prone PCR to yield randommutations after subsequent homologous recombination of mutagenizedcoding sequence and linearized vector after co-electroporation. Mutantsthat displayed higher yeast surface levels, and thus presumptiveenhanced stability, were identified by flow cytometry sorting.

[0050] The unstable class MHC protein L^(d) was also cloned as an AGA2fusion in single chain format (AGA2-HA-L^(d)-β2m-c-myc). L^(d) wasdetected on the yeast cell surface with an α3-specific antibody, butproperly folded α1/α2 domains were not detected with anti-L^(d)antibody, suggesting that stabilized variants of L^(d) can be engineeredby directed evolution and flow sorting for improved yeast surfaceexpression.

[0051] The present inventors have now succeeded in isolating stabilizedmutants of the class I molecule L^(d). The L^(d) molecule has been shownto be relatively unstable. When expressed in the yeast display system,only the α3 domain appears to be folded properly on the surface (FIG.16). The displayed protein is not recognized by a conformation-specificα2 antibody (30-5-7) nor is the QL9/L^(d) complex recognized by thehigh-affinity T cell receptor called m6 (FIG. 16). To isolate stabilizedL^(d), a library of random mutants was expressed in yeast, and thelibrary was selected with anti-L^(d) antibody 30-5-7; see FIG. 17 andits description. Various mutants were isolated. One mutant (30.8) isshown in FIG. 17. Mutant 30.8 bound to both the α2-specific antibody andthe QL9/L^(d) (30.8) complex bound to the high affinity TCR m6,reflecting increased stability over its wild type parent protein.

[0052] Another application of the yeast display to technology is toidentify peptides that bind directly to MHC class I proteins.Serendipitously, we isolated a peptide that bound to the K^(b) moleculeeven though the peptide was fused to the C-terminus of the AGA2 protein.This finding was completely unexpected. Previously, only peptides of 8to 10 amino acids had been shown to bind to class I MHC products. Thisrestriction in length was thought to be due to the need for both the N-and C-termini to bind within pockets of the class I molecule, therebycontributing binding energy to the interaction. AGA2-fusion peptideswere isolated by screening with a dEV8/K^(b) tetramer. A carboxyterminal peptide sequence HYSPFRQLA (SEQ ID NO:37) that hadK^(b)-binding consensus anchor residues at positions P5 and P8 wasisolated (FIG. 18). Yeast displaying the AGA2-fusion with the sequenceHYSPFRQLA (SEQ ID NO:37) at the C-terminus bound to dEV8/K^(b) tetramers(FIG. 19). This binding was a consequence of the relative instability ofdEV8 binding to K^(b). This instability apparently allows the peptidedEV8 to dissociate, and the surface-bound AGA2-fusion peptide binds tothe free K^(b). Without wishing to be bound by any particular theory, webelieve that the proline at position P4 allows the peptide to exit theK^(b)-site at the residue amino terminal to the P5 anchor. This issupported by the finding that an alanine substitution mutation at thisposition has significantly reduced binding (FIG. 19). These findingsshow that it is possible to create a library of fused peptides (to AGA2)and that this library can be screened for K^(b)-binding (FIG. 20). Thus,the yeast display system can be used to identify novel class I-bindingpeptides.

[0053] In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given to such terms,the following definitions are provided.

[0054] A coding sequence is the part of a gene or cDNA which codes forthe amino acid sequence of a protein, or for a functional RNA such as atRNA or rRNA.

[0055] Complement or complementary sequence means a sequence ofnucleotides which forms a hydrogen-bonded duplex with another sequenceof nucleotides according to Watson-Crick base-pairing rules. Forexample, the complementary base sequence for 5′-AAGGCT-3′ is3′-TTCCGA-5′.

[0056] Downstream means on the 3′ side of any site in DNA or RNA.

[0057] Expression refers to the transcription of a gene into structuralRNA (rRNA, tRNA) or messenger RNA (mRNA) and subsequent translation of amRNA into a protein.

[0058] An amino acid sequence that is functionally equivalent to aspecifically exemplified MHC protein sequence is an amino acid sequencethat has been modified by single or multiple amino acid substitutions,by addition and/or deletion of amino acids, or where one or more aminoacids have been chemically modified, but which nevertheless retains thebinding specificity and high affinity binding activity of a cell-boundor a soluble MHC protein of the present invention. Functionallyequivalent nucleotide sequences are those that encode polypeptideshaving substantially the same biological activity as a specificallyexemplified cell-bound or soluble MHC protein. In the context of thepresent invention, a soluble MHC protein is lacks the portions of anative cell-bound MHC and is stable in solution (i.e., it does notgenerally aggregate in solution when handled as described herein andunder standard conditions for protein solutions).

[0059] Two nucleic acid sequences are heterologous to one another if thesequences are derived from separate organisms, whether or not suchorganisms are of different species, as long as the sequences do notnaturally occur together in the same arrangement in the same organism.

[0060] Homology refers to the extent of identity between two nucleotideor amino acid sequences.

[0061] Isolated means altered by the hand of man from the natural state.If an “isolated” composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not isolated, but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is isolated, as theterm is employed herein.

[0062] A linker region is an amino acid sequence that operably links twofunctional or structural domains of a protein.

[0063] A nucleic acid construct is a nucleic acid molecule which isisolated from a naturally occurring gene or which has been modified tocontain segments of nucleic acid which are combined and juxtaposed in amanner which would not otherwise exist in nature.

[0064] Nucleic acid molecule means a single- or double-stranded linearpolynucleotide containing either deoxyribonucleotides or ribonucleotidesthat are linked by 3′-5′-phosphodiester bonds.

[0065] Two DNA sequences are operably linked if the nature of thelinkage does not interfere with the ability of the sequences to effecttheir normal functions relative to each other. For instance, a promoterregion would be operably linked to a coding sequence if the promoterwere capable of effecting transcription of that coding sequence.

[0066] A polypeptide is a linear polymer of amino acids that are linkedby peptide bonds.

[0067] Promoter means a cis-acting DNA sequence, generally 80-120 basepairs long and located upstream of the initiation site of a gene, towhich RNA polymerase binds and initiates correct transcription. Therecan be associated additional transcription regulatory sequences whichprovide on/off regulation of transcription and/or which enhance(increase) expression of the downstream coding sequence.

[0068] A recombinant nucleic acid molecule, for instance a recombinantDNA molecule, is a novel nucleic acid sequence formed in vitro throughthe ligation of two or more nonhomologous DNA molecules (for example arecombinant plasmid containing one or more inserts of foreign DNA clonedinto at least one cloning site), through PCR technology or by directedhomologous recombination, e.g. by co-transformation of two or more DNAmolecules having at least regions of limited sequence identity to allowfor homologous recombination with the transformed cell.

[0069] Transformation means the directed modification of the genome of acell by the external application of purified recombinant DNA fromanother cell of different genotype, leading to its uptake andintegration into the subject cell's genome. In bacteria, the recombinantDNA is not typically integrated into the bacterial chromosome, butinstead replicates autonomously as a plasmid.

[0070] Upstream means on the 5′ side of any site in DNA or RNA.

[0071] A vector is a nucleic acid molecule that is able to replicateautonomously in a host cell and can accept foreign DNA. A vector carriesat least one origin of replication functional in at least one type ofcell, one or more unique recognition sites for restriction endonucleaseswhich can be used for the insertion of foreign DNA, and usuallyselectable markers such as genes coding for antibiotic resistance, andoften recognition sequences (e.g. promoter) for the expression of theinserted DNA. Common vectors include plasmid vectors and phage vectors.There can be more than one origin of replication to allow forreplication and maintenance in more than one type of cell (e.g.,separate origins of replication functional in yeast and Escherichiacoli, respectively).

[0072] A virus infected cell is a cell in which a virus is replicating.Typically, a virus infected cell displays at least one antigen on itssurface which is characteristic of the virus infection process. Such anantigen can be the target of recognition by the immune system andsubsequent killing of the cell.

[0073] A pathogen infected cell is a human or animal cell in which anintracellular parasite (bacterial, fungal or protozoan) is surviving orreproducing. Typically a pathogen infected cell displays at least oneantigen on its surface which is characteristic of the infection, andthis antigen can be the target of immune recognition and targeting forkilling of the infected cell.

[0074] The role of proteins encoded by the major histocompatibilitycomplex (MHC proteins) have been known for nearly twenty years. MHCproteins are expressed by every individual, and they function asantigen-presenting molecules. Each MHC protein can bind to a variety ofdifferent small peptides (8 to 20 amino acids).

[0075] Recently it was demonstrated that a scTCR (Vβ-linker-Vα) could bedisplayed on the surface of yeast [Kieke et al. (1999) Proc. Natl. Acad.Sci. USA 96:5651-5656], in the yeast display system that has provensuccessful in antibody engineering [Boder and Wittrup (1997) Nat.Biotech. 15: 553-557.; Kieke et al. (1999) supra]. In addition, it wasshown that mutations that increased the surface levels of the TCR alsoincreased the sability of the TCR in solution [Shusta et al. (1999) J.Mol. Biol. 292:949-956]. Thus, yeast surface display can now be used toisolate proteins that exhibit greater stability.

[0076] It will be appreciated by those of skill in the art that, due tothe degeneracy of the genetic code, numerous functionally equivalentnucleotide sequences encode the same amino acid sequence of an improvedClass I MHC protein or class I MHC/peptide complex.

[0077] Additionally, those of skill in the art, through standardmutagenesis techniques, in conjunction with the antigen-finding activityassays described herein, can obtain altered MHC class I proteinsequences and test them for the expression of polypeptides havingparticular binding activity. Useful mutagenesis techniques known in theart include, without limitation, oligonucleotide-directed mutagenesis,region-specific mutagenesis, linker-scanning mutagenesis, andsite-directed mutagenesis by PCR [see e.g. Sambrook et al. (1989) videinfra, and Ausubel et al. (1999) vide infra].

[0078] In obtaining variant MHC coding sequences, those of ordinaryskill in the art will recognize that MHC-derived proteins can bemodified by certain amino acid substitutions, additions, deletions, andpost-translational modifications, without loss or reduction ofbiological activity. In particular, it is well-known that conservativeamino acid substitutions, that is, substitution of one amino acid foranother amino acid of similar size, charge, polarity and conformation,are unlikely to significantly alter protein function. The 20 standardamino acids that are the constituents of proteins can be broadlycategorized into four groups of conservative amino acids as follows: thenonpolar (hydrophobic) group includes alanine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan and valine; the polar(uncharged, neutral) group includes asparagine, cysteine, glutamine,glycine, serine, threonine and tyrosine; the positively charged (basic)group contains arginine, histidine and lysine; and the negativelycharged (acidic) group contains aspartic acid and glutamic acid.Substitution in a protein of one amino acid for another within the samegroup is unlikely to have an adverse effect on the biological activityof the protein.

[0079] Homology between nucleotide sequences can be determined by DNAhybridization analysis, wherein the stability of the double-stranded DNAhybrid is dependent on the extent of base pairing that occurs.Conditions of high temperature and/or low salt content reduce thestability of the hybrid, and can be varied to prevent annealing ofsequences having less than a selected degree of homology. For instance,for sequences with about 55% G-C content, hybridization and washconditions of 40-50 C, 6× SSC (sodium chloride/sodium citrate buffer)and 0.1% SDS (sodium dodecyl sulfate) indicate about 60-70% homology,hybridization and wash conditions of 50-65 EC, 1× SSC and 0.1% SDSindicate about 82-97% homology, and hybridization and wash conditions of52 C, 0.1× SSC and 0.1% SDS indicate about 99 -100% homology. A widerange of computer programs for comparing nucleotide and amino acidsequences (and measuring the degree of homology) are also available, anda list providing sources of both commercially available and freesoftware is found in Ausubel et al. (1999). Readily available sequencecomparison and multiple sequence alignment algorithms are, respectively,the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1997)and ClustalW programs. BLAST is available on the Internet athttp://www.ncbi.nlm.nih.gov and a version of ClustalW is availableathttp://www2.ebi.ac.uk.

[0080] Industrial strains of microorganisms (e.g., Aspergillus niger,Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae, Trichodermareesei, Mucor miehei, Kluyveromyces lactis, Pichiapastoris,Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis orBacillus licheniformis) or plant species (e.g., canola, soybean, corn,potato, barley, rye, wheat) may be used as host cells for therecombinant production of the mutant MHC proteins of the presentinvention. As the first step in the heterologous expression of a highaffinity MHC protein or soluble protein, an expression construct isassembled to include the MHC or soluble MHC coding sequence and controlsequences such as promoters, enhancers and terminators. Other sequencessuch as signal sequences and selectable markers may also be included. Toachieve extracellular expression of a soluble MHC polypeptide, theexpression construct may include a secretory signal sequence. The signalsequence is not included on the expression construct if cytoplasmicexpression is desired. The promoter and signal sequence are functionalin the host cell and provide for expression and secretion of the MHC orsoluble MHC protein. Transcriptional terminators are included to ensureefficient transcription. Ancillary sequences enhancing expression orprotein purification may also be included in the expression construct.

[0081] Various promoters (transcriptional initiation regulatory region)may be used according to the invention. The selection of the appropriatepromoter is dependent upon the proposed expression host. Promoters fromheterologous sources may be used as long as they are functional in thechosen host.

[0082] Promoter selection is also dependent upon the desired efficiencyand level of peptide or protein production. Inducible promoters such tacare often employed in order to dramatically increase the level ofprotein expression E. coli. Overexpression of proteins may be harmful tothe host cells. Consequently, host cell growth may be limited. The useof inducible promoter systems allows the host cells to be cultivated toacceptable densities prior to induction of gene expression, therebyfacilitating higher product yields.

[0083] Various signal sequences may be used according to the invention.A signal sequence which is homologous to the MHC coding sequence may beused. Alternatively, a signal sequence which has been selected ordesigned for efficient secretion and processing in the expression hostmay also be used. For example, suitable signal sequence/host cell pairsinclude the B. subtilis sacB signal sequence for secretion in B.subtilis, and the Saccharomyces cerevisiae α-mating factor or P.pastoris acid phosphatase phoi signal sequences for P. pastorissecretion. The signal sequence may be joined directly through thesequence encoding the signal peptidase cleavage site to the proteincoding sequence, or through a short nucleotide bridge consisting ofusually fewer than ten codons, where the bridge ensures correct readingframe of the downstream TCR sequence.

[0084] Elements for enhancing transcription and translation have beenidentified for eukaryotic protein expression systems. For example,positioning the cauliflower mosaic virus (CaMV) promoter 1000 bp oneither side of a heterologous promoter may elevate transcriptionallevels by 10- to 400-fold in plant cells. The expression constructshould also include the appropriate translational initiation sequences.Modification of the expression construct to include a Kozak consensussequence for proper translational initiation may increase the level oftranslation by 10 fold.

[0085] A selective marker is often employed, which may be part of theexpression construct or separate from it (e.g., carried by theexpression vector), so that the marker may integrate at a site differentfrom the gene of interest. Examples include markers that conferresistance to antibiotics (e.g., bla confers resistance to ampicillinfor E. coli host cells, nptII confers kanamycin resistance to a widevariety of prokaryotic and eukaryotic cells) or that permit the host togrow on minimal medium (e.g., HIS4 enables P. pastoris or His-S.cerevisiae to grow in the absence of histidine). The selectable markerhas its own transcriptional and translational initiation and terminationregulatory regions to allow for independent expression of the marker. Ifantibiotic resistance is employed as a marker, the concentration of theantibiotic for selection will vary depending upon the antibiotic,generally ranging from 10 to 600 μg of the antibiotic/mL of medium.

[0086] The expression construct is assembled by employing knownrecombinant DNA techniques (Sambrook et al., 1989; Ausubel et al.,1999). Restriction enzyme digestion and ligation are the basic stepsemployed to join two fragments of DNA. The ends of the DNA fragment mayrequire modification prior to ligation, and this may be accomplished byfilling in overhangs, deleting terminal portions of the fragment(s) withnucleases (e.g., ExoIII), site directed mutagenesis, or by adding newbase pairs by PCR. Polylinkers and adaptors may be employed tofacilitate joining of selected fragments. The expression construct istypically assembled in stages employing rounds of restriction, ligation,and transformation of E. coli. Homlogons recombination strategies can beused with co-electroporation of linear DNAs into yeast (see FIG. 9).Numerous cloning vectors suitable for construction of the expressionconstruct are known in the art (λZAP and pBLUESCRIPT SK-1, Stratagene,LaJolla, Calif.; pET, Novagen Inc., Madison, Wis.; cited in Ausubel etal., 1999) and the particular choice is not critical to the invention.The selection of cloning vector will be influenced by the gene transfersystem selected for introduction of the expression construct into thehost cell. At the end of each stage, the resulting construct may beanalyzed by restriction, DNA sequence, hybridization and PCR analyses.

[0087] The expression construct may be transformed into the host as thecloning vector construct, either linear or circular, or may be removedfrom the cloning vector and used as is or introduced onto a deliveryvector. The delivery vector facilitates the introduction and maintenanceof the expression construct in the selected host cell type. Theexpression construct is introduced into the host cells by any of anumber of known gene transfer systems (e.g., natural competence,chemically mediated transformation, protoplast transformation,electroporation, biolistic transformation, transfection, or conjugation)(Ausubel et al., 1999; Sambrook et al., 1989). The gene transfer systemselected depends upon the host cells and vector systems used.

[0088] For instance, the expression construct can be introduced into S.cerevisiae cells by protoplast transformation or electroporation.Electroporation of S. cerevisiae is readily accomplished, and yieldstransformation efficiencies comparable to spheroplast transformation.

[0089] Monoclonal or polyclonal antibodies, preferably monoclonal,specifically reacting with a protein of interest may be made by methodsknown in the art. See, e.g., Harlow and Lane (1988) Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratories; Goding (1986)Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press,New York; and Ausubel et al. (1999) Current Protocols in MolecularBiology, John Wiley & Sons, Inc., New York.

[0090] MHC/peptide complexes of improved stability in cell-bound orsoluble form which are characteristic of a particular neoplasticcondition (cancer, tumor, or the like) or a particular virus infectedcell or pathogen infected cell are useful, for example, as agonists ofthe immune system so that the neoplastic cell, virus infected cell orpathogen infected cell is more efficiently targeted for removal by Tcells of the immune system. The improved MHC/p complexes can be labeledby joining, either covalently or noncovalently, a substance whichprovides a detectable signal. Suitable labels include but are notlimited to radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent agents, chemiluminescent agents, magnetic particles and thelike. Additionally the MHC protein of the present invention can becoupled to a ligand for a second binding molecules: for example, the MHCprotein can be biotinylated. Detection of the MHC protein or complex canbe effected by binding of a detectable streptavidin (a streptavidin towhich a fluorescent, radioactive, chemiluminescent, or other detectablemolecule is attached or to which an enzyme for which there is achromophoric substrate available). United States patents describing theuse of such labels and/or toxic compounds to be covalently bound to thescTCR protein include but are not limited to U.S. Pat. Nos. 3,817,837;3,850,752; 3,927,193; 3,939,350; 3,996,345; 4,277,437; 4,275,149;4,331,647; 4,348,376; 4,361,544; 4,468,457; 4,444,744; 4,640,561;4,366,241; RE 35,500; 5,299,253; 5,101,827; 5,059,413. Labeled MHCproteins or complexes can be detected using a monitoring device ormethod appropriate to the label used. Fluorescence microscopy orfluorescence activated cell sorting can be used where the label is afluorescent moiety, and where the label is a radionuclide, gammacounting, autoradiography or liquid scintillation counting, for example,can be used with the proviso that the method is appropriate to thesample being analyzed and the radionuclide used. In addition, there canbe secondary detection molecules or particle employed where there is adetectable molecule or particle which recognized the portion of the MHCprotein which is not part of the binding site for the cognate TCR orother ligand or other ligand in the absence of a component as notedherein. The art knows useful compounds for diagnostic imaging in situ;see, e.g., U.S. Pat. No. 5,101,827; 5,059,413. Radionuclides useful fortherapy and/or imaging in vivo include ¹¹¹Indium, ⁹⁷Rubidium, ¹²⁵Iodine,¹³¹Iodine, ¹²³Iodine, ⁶⁷Gallium, ⁹⁹Technetium. Toxins include diphtheriatoxin, ricin and castor bean toxin, among others, with the proviso thatonce the TCR-toxin complex is bound to the cell, the toxic moiety isinternalized so that it can exert its cytotoxic effect. Immunotoxintechnology is well known to the art, and suitable toxic moleculesinclude, without limitation, chemotherapeutic drugs such as vindesine,antifolates, e.g. methotrexate, cisplatin, mitomycin, anthrocyclinessuch as daunomycin, daunorubicin or adriamycin, and cytotoxic proteinssuch as ribosome inactivating proteins (e.g., diphtheria toxin, pokeweedantiviral protein, abrin, ricin, pseudomonas exotoxin A or theirrecombinant derivatives. See, generally, e.g., Olsnes and Pihl (1982)Pharmac. Ther. 25:355-381 and Monoclonal Antibodies for Cancer Detectionand Therapy, Eds. Baldwin and Byers, pp. 159-179, Academic Press, 1985.

[0091] Table, high affinity MHC proteins specific for a particularligand, e.g., a particular peptide, protein or cell type, are useful indiagnosing animals, including humans, believed to be suffering from adisease associated with the particular pMHC. The MHC molecules of thepresent invention are useful for detecting T cells that are specific foressentially any antigen including, but not limited to, those associatedwith a neoplastic condition, an abnormal protein, or an infection orinfestation with a bacterium, a fungus, a virus, a protozoan, a yeast, anematode or other parasite. Stable, high affinity MHC proteins specificfor a particular ligand can also be used to induce the activity of Tcells against antigens if desirable. For example, there are manypeptides that have been associated with neoplastic cells, abnormalproteins, bacteria, fungi, a viruses, and protozoans whereby saidpeptides bind to a Class I MHC protein. Stable, high affinity MHCproteins in complex with these peptides could serve as vaccines againstthe diseases by inducing T cell activity.

[0092] The high affinity MHC compositions can be formulated by any ofthe means known in the art. They can be typically prepared asinjectables, especially for intravenous, intraperitoneal or synovialadministration (with the route determined by the particular disease) oras formulations for intranasal or oral administration, either as liquidsolutions or suspensions. Solid forms suitable for solution in, orsuspension in, liquid prior to injection or other administration mayalso be prepared. The preparation may also, for example, be emulsified,or the protein(s)/peptide(s) encapsulated in liposomes.

[0093] The active ingredients are often mixed with excipients orcarriers which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients include but are not limited towater, saline, dextrose, glycerol, ethanol, or the like and combinationsthereof. The concentration of the MHC protein in injectable, aerosol ornasal formulations is usually in the range of 0.05 to 5 mg/ml. Similardosages can be administered to other mucosal surfaces.

[0094] In addition, if desired, vaccines may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include but are not limitedto: aluminum hydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy) -ethylamine (CGP19835A, referred to as MTP-PE); and RIBI, which contains threecomponents extracted from bacteria: monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. Such additional formulations and modes of administration asare known in the art may also be used.

[0095] The stable high affinity MHC proteins of the present inventionand/or pMHC-binding fragments having primary structure similar (morethan 90% identity) to the high affinity MHC proteins and which maintainthe high affinity for the cognate ligand may be formulated into vaccinesas neutral or salt forms. Pharmaceutically acceptable salts include butare not limited to the acid addition salts (formed with free aminogroups of the peptide) which are formed with inorganic acids, e.g.,hydrochloric acid or phosphoric acids; and organic acids, e.g., acetic,oxalic, tartaric, or maleic acid. Salts formed with the free carboxylgroups may also be derived from inorganic bases, e.g., sodium,potassium, ammonium, calcium, or ferric hydroxides, and organic bases,e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine,and procaine. Alternatively, these stable high affinity MHC proteins canbe used as antagonists of an interaction between endogenous MHC proteinsof similar specificity and the cognate TCR cells.

[0096] High affinity MHC proteins or complexes for therapeutic use,e.g., those conjugated to cytotoxic compounds are administered in amanner compatible with the dosage formulation, and in such amount andmanner as are prophylactically and/or therapeutically effective,according to what is known to the art. The quantity to be administered,which is generally in the range of about 100 to 20,000 μg of protein perdose, more generally in the range of about 1000 to 10,000 μg of proteinper dose. Similar compositions can be administered in similar ways usinglabeled high affinity MHC proteins for use in imaging, for example, todetect deleterious cytotoxic T cells that are involved in autoimmuneattacks and containing the cognate pMHCs. Precise amounts of the activeingredient required to be administered may depend on the judgment of thephysician or veterinarian and may be peculiar to each individual, butsuch a determination is well within the skill of such a practitioner.

[0097] The vaccine or other immunogenic composition may be given in asingle dose; two dose schedule, for example two to eight weeks apart; ora multiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may include 1 to 10 or more separatedoses, followed by other doses administered at subsequent time intervalsas required to maintain and/or reinforce the immune response, e.g., at 1to 4 months for a second dose, and if needed, a subsequent dose(s) afterseveral months. Humans (or other animals) immunized with theretrovirus-like particles of the present invention are protected frominfection by the cognate retrovirus.

[0098] Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993)Meth. Enzmol. 218, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al.(eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.)Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oldand Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkeley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) NucleicAcidHybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York. Abbreviations and nomenclature, where employed, are deemedstandard in the field and commonly used in professional journals such asthose cited herein.

[0099] All references cited in the present application are incorporatedby reference herein to supplement the disclosure and experimentalprocedures provided in the present Specification to the extent thatthere is no inconsistency with the present disclosure.

[0100] The following examples are provided for illustrative purposes,and are not intended to limit the scope of the invention as claimedherein. Any variations in the exemplified articles and/or methods whichoccur to the skilled artisan are intended to fall within the scope ofthe present invention.

EXAMPLES Example 1 Fusion of the MHC α Chain to the β2m in the YeastDisplay Vector pCT302

[0101] The mouse MHC K^(b) α chain was fused to the mouse β2m [Mottez etal. (1995) J. Exp. Med. 181(2), 493-502]. The K^(b) gene was PCRamplified using primers with Nhe I and Afl II restriction sites. The 5′primer contained a 30 bp linker upstream of the K^(b) gene and the 3′primer contained a 45 bp connecting linker (underlined) downstream ofthe gene (5′ CAA TGG CTA GCG GTG GAC TTA AGG GTG GAC CAG GTG GAG GTT CAGGAG GTG GAG GCC CAC ACT CGC TGA GGT ATT TCG T 3′, SEQ ID NO:7; and 5′TGA ACC TCC GCC TCC TGA TCC ACC GCC ACC TGA ACC TAT TCC ACC CTC CCA TCTCAG GGT GAG GGG CTC AGG 3′, SEQ ID NO. 8). The β2m gene was PCRamplified with the 5′ primer containing the overlapping 45 bp linker(underlined) upstream and the 3′ primer containing a c-myc epitope tagand unique Xho I site downstream of the β2m (5′ GGT GGA ATA GGT TCA GGTGGC GGT GGA TCA GGA GGC GGA GGT TCA ATC CAG AAA ACC CCT CAA ATT CAA GTAT 3′, SEQ ID NO:9, and 5 ′GTT CCC TCG AGC TAT TAC AAG TCT TCT TCA GAAATA AGC TTT TGT TCC ATG TCT CGA TCC CAG TAG ACG GT 3′, SEQ ID NO:10).Using PCR “sewing” of the overlapping linker [Davis et al. (1992)Biotechnology 9(2), 165-169], an amplified K^(b)/β2m fusion wasgenerated using both the K^(b) and β2m PCR products, primers 1 and 4 andTaqPlus Precision PCR System (Stratagene, La Jolla, Calif.). TheL^(d)/β2m MHC gene was fused and PCR amplified in an analogous manner.The K^(b)/β2m and L^(d)/β2m PCR products were digested with Nhe I/Xho Iand ligated into the yeast surface display vector pCT302 containing anine-residue epitope tag (HA) and the AGA2 open reading frame downstreamof the inducible GALL promoter [Boder and Wittrup (1997) supra].

Example 2 Peptide/MHC Construction

[0102] The AGA2 gene was cloned at the COOH terminus of thepeptide/K^(b)/β2m gene in order to allow a free NH₂ terminus of thepeptide. First, the K^(b)/β2m signal sequence was PCR amplified withEcoRI and Nhe I restriction sites upstream and downstream of the signalsequence (SS) (5′ TCT CAA GAA TTC TAC TTC ATA CAT TTT 3′, SEQ ID NO:11;and

[0103] 5′ GTA TCT GCT AGC TGC TAA AAC TGA AGC 3′, SEQ ID NO:12). The SSPCR product was digested Nhe I/Eco RI and ligated into the Nhe I/Eco RIdigested pCT302 vector. The resulting plasmid contained the SS but notthe AGA2 gene. Secondly, a SIYRYYGL (SEQ ID NO:1) (SIYR)/K^(b)/β2m PCRproduct was generated with a SIYR encoded primer (underlined) (5′ ATACTA GCT AGC TTG GAT AAA AGG TCT ATT TAT AGA TAT TAT GGT TTG CTT AAG GGTGGA CCA GGT GGA GGT 3′, SEQ ID NO:13; and 5′ CAA TCC AGA TCT TTA CTA ATGCAA GTC TTC TTC AGA AAT AAG 3′, SEQ ID

[0104] NO:14). Nhe I and Nde I restriction sites are located in theupstream and downstream primers respectively. The SIYR/K^(b)/β2m PCRproduct was digested with Nhe I and Nde I and ligated into the Nhe I/NdeI digested pCT302 SS vector (now called SS-SIYR K^(b) pCT302). Finally,AGA2 was PCR amplified (5′ GGA TAT CAT ATG CAG GAA CTG ACA ACT ATA3′,SEQ ID NO:15, and 5′ ATT TGC AGA TCT CGA GTT ACT AAG CGT AGT CTG GAA CGTCGT A 3′, SEQ ID NO:16), digested with Nde I and Xho I and ligated intothe Nde I/Xhol digested SS-SIYR K^(b) pCT302. The resulting constructcontained the following order of genes in the pCT302 backbone: SS-SIYRK^(b)/β2m-AGA2.

Example 3 OVA and dEV8 Peptide Loading

[0105] The sense and anti-sense oligonucleotide sequences for both OVA(SIINFEKL, SEQ ID NO:2) and dEV8 (EQYKFYSV, SEQ ID NO:5) with a Nhe Iand Afl II site upstream and downstream respectively were eachphosphorylated with T4 DNA kinase (OVA 5° CT AGC TTG GAT AAA AGG AGC ATCATC AAT TTT GAA AAG CTT C3′, SEQ ID NO:17; and 5′TT AAG AAG CTT TTC AAAATT GAT GAT GCT CCT TTT ATC CAA G3′, SEQ ID NO:18; dEV8 5° CT AGC TTGGAT AAA AGG GAA CAA TAC AAA TTC TAC TCA GTT C3′, SEQ ID NO:19; and 5′TTAAG AAC TGA GTA GAA TTT GTA TTG TTC CCT TTT ATC CAA G3′, SEQ ID NO:20).The sense and anti-sense phosphorylated oligonucleotides were mixed (400pmol), heated at 100° C. for 1 min and cooled slowly. Phosphorylatedpeptide cassettes were ligated into Nhe I/Afl II digested SIYRK^(b)/β2mpCT302. Ligation reactions were transformed into DHIOB electrocompetentE. coli cells and plated on LB/amp and incubated for 15 hrs at 37 C.Transformants were screened, and positive clones were sequenced. Theresulting plasmids contained OVA and dEV8 tethered to K^(b)/β2m.

Example 4 Transformation into Yeast

[0106] The resultant MHC nucleotide constructs were transformed by thelithium acetate (LiAc) transformation method [Geitz et al. (1995) Yeast11, 355-360] into the S. cerevisiae strain BJ5465 (α ura3-52 trp1 leu2Δ1his3Δ200pep4::HIS2prbΔ1.6 can1 GAL; Yeast Genetic Stock Center,Berkeley, Calif.) containing a chromosomally integrated AGA1 codingsequence expressed under the control of the GAL1 promoter (strainEBY100; Boder and Wittrup (1997) supra).

Example 5 Induction and Detection of MHC on the Yeast Surface

[0107] Yeast cells transformed with pCT302/MHC plasmid constructs weregrown overnight at 30 C with shaking in 2 mL selective glucose mediumSD-CAA (glucose 2 wt %, Difco yeast nitrogen base 0.67 wt %, casaminoacids 0.5 wt %). After 18-24 hours, recombinant AGAL+AGA2-MHC Iexpression was induced at 20° C. with shaking in 5 mL selectivegalactose medium (SG-CAA, where 2% galactose replaces the glucose inSD-CAA). Cultures were harvested after 24-48 hours (1-2 doublings) bycentrifugation, washed with PBS (10 mM NaPO₄, 150 mM NaCl, pH 7.3)containing 0.5% bovine serum albumin and incubated 45 minutes on icewith 25 μL of an anti-MHC antibody, anti-c-myc Mab 9E10 (1:100 dilutionof raw ascites fluid; Berkeley Antibody Co., Richmond, Calif.), oranti-HA Mab 12CA5 (10 μg/ml. Boehringer Mannheim, Indianapolis, Ind.).Cells were washed with PBS and incubated 30 minutes on ice with eitherFITC-labeled F(ab′)₂ goat anti-mouse IgG (1:50; Kirkegaard and PerryLabs, Inc., Gaithersburg, Md.) or a streptavidin-phycoerythrin (SA-PE)conjugate (1:200; PharMingen, San Diego, Calif.). Labeled yeast cellswere analyzed on a Coulter Epics XL flow cytometer. Data for10,000-20,000 events were collected, and the population was gatedaccording to light scatter (size) to prevent analysis of cell clumps.

Example 6 Random Mutagenesis of dEV8/K^(b)/β2m and SIYR/K^(b)/β2m

[0108] dEV8/K^(b)/β2m and SIYR/K^(b)/β2m genes were randomly mutagenizedusing a PCR error prone technique. dEV8/K^(b)/β2m and SIYR/K^(b)/β2mwere PCR amplified using vector specific primers at least 50 bp upstreamand downstream of each gene with PCR conditions that cause randommutations to be inserted by Taq polymerase (GIBCO/BRL,Invitrogen,Carelsbad, Calif.). At a Mn:Mg ratio of about 0.16: 1, thepolymerase is more susceptible to inserting a random nucleotide duringelongation. Using homologous recombination [Raymond et al. (1999)Biotechniques 26(1): 134-138, 140-141], the dEV8/K^(b)/β2m andSIYR/K^(b)/β2m pCT302 vectors digested with Nhe and NdeI were combinedwith the error prone PCR products and electroporated (Bio-RadGene-Pulser II, 1.5V, 25μF, 0.2cm gene pulser cuvettes) into 40 μl ofelectrocompetent S. cerevisiae cells (BJ5465, strain EBY100). Theresulting transformations (separately for the two chimeric genes) arepooled, and a dilution is plated on SD-CAA plates. Plates are incubatedat 30 C for 3 days, and the library size is tabulated.

Example 7 Cell Sorting

[0109] The yeast library was grown in SD-CAA (2% dextrose, 0.67% yeastnitrogen base, 1% Casamino acids (Difco, Detroit, Mich.)) at 30 C to anOD₆₀₀=4.0. To induce surface scTCR expression, yeast cells were pelletedby centrifugation, resuspended to an OD₆₀₀=1.0 in SG-CAA (2% galactose,0.67% yeast nitrogen base, 1% casamino acids), and incubated at 20 C for48 hr. In general, 10⁷ cells/tube were incubated on ice for 1 hr with 50μl of biotinylated anti-K^(b) antibody (B.8.24.3) diluted in phosphatebuffered saline (pH 7.4) supplemented with 0.5 mg/ml BSA (PBS-BSA).After incubation, cells were washed and labeled for 30 min with SA:PE inPBS-BSA. Yeast cells were then washed and resuspended in PBS-BSAimmediately prior to sorting. Cells exhibiting the highest fluorescencewere isolated by FACS sorting with a Coulter 753 bench. After isolation,sorted cells were expanded in SD-CAA and induced in SG-CAA forsubsequent rounds of selection. Three sequential sorts were performedfor each mutant preparation with increasingly dilute anti-K^(b). Thepercentages of total cells isolated from each sort were 1.0, 0.25 and 0.1%, respectively. Aliquots of the third sorts were plated on SD-CAA toisolate individual clones, which were then analyzed by flow cytometryusing a Coulter Epics XL instrument. The clones with the highestfluorescence have their DNA rescued with the Zymoprep Yeast PlasmidMiniprep Kit (Zymogen Research, Orange, Calif.). The DNA isretransformed into DH10 Belectrocompetent E. coli, mini prepped andsubmitted for sequencing. The sequences are analyzed, and the mutationsare located. TABLE 1 Comparison of Properties of H-2K^(b) , H-2D^(b) andH-2 L^(d) Proteins H-2K^(b) H-2D^(b) H-2L^(d) Heavy chain-B2m vdw 17 247 H bonds 7 13 10 alpha 1/alpha2: B2m vdw 13 16 4 H bonds 4 5 2 alpha 3:B2m vdw 4 8 3 H bonds 3 8 8

[0110] TABLE 2 Various Single-Chain Class I MHC Constructions Cloned inpCT3O2 Yeast Display Vector Nucleic Acid Sequence 1:SS-AGA2-K^(b)/β2m-c-myc (SEQ ID NO:22)ATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTAGCACAGGAACTGACAACTATATGCGAGCAAATCCCCTCACCAACTTTAGAATCGACGCCGTACTCTTTGTCAACGACTACTATTTTGGCCAACGGGAAGGCAATGCAAGGAGTTTTTGAATATTACAAATCAGTAACGTTTGTCAGTAATTGCGGTTCTCACCCCTCAACAACTAGCAAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGGACAATAGCTCGACGATTGAAGGTAGATACCCATACGACGTTCCAGACTACGCTCTGCAGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCGGTGGACTTAAGGGTGGACCAGGTGGAGGTTCAGGAGGTGGAGGCCCACACTCGCTGAGGTATTTCGTCACCGCCGTGTCCCGGCCCGGCCTCGGGGAGCCCCGGTACATGGAAGTCGGCTACGTGGACGACACGGAGTTCGTGCGCTTCGACAGCGACGCGGAGAATCCGAGATATGAGCCGCGGGCGCGGTGGATGGAGCAGGAGGGGCCCGAGTATTGGGAGCGGGAGACACAGAAAGCCAAGGGCAATGAGCAGAGTTTCCGAGTGGACCTGAGGACCCTGCTCGGCTACTACAACCAGAGCAAGGGCGGCTCTCACACTATTCAGGTGATCTCTGGCTGTGAAGTGGGGTCCGACGGGCGACTCCTCCGCGGGTACCAGCAGTACGCCTACGACGGCTGCGATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACATGGCGGCGCTGATCACCAAACACAAGTGGGAGCAGGCTGGTGAAGCAGAGAGACTCAGGGCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCGCAGATACCTGAAGAACGGGAACGCGACGCTGCTGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTGAAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGACATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATGGAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGTGTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGGGTGGAATAGGTTCAGGTGGCGGTGGATCAGGAGGCGGAGGTTCAATCCAGAAAACCCCTCAAATTCAAGTATACTCACGCCACCCACCGGAGAATGGGAAGCCGAACATACTGAACTGCTACGTAACACAGTTCCACCCGCCTCACATTGAAATCCAAATGCTGAAGAACGGGAAAAAAATTCCTAAAGTAGAGATGTCAGATATGTCCTTCAGCAAGGACTGGTCTTTCTATATCCTGGCTCACACTGAATTCACCCCCACTGAGACTGATACATACGCCTGCAGAGTTAAGCATGACAGTATGGCCGAGCCCAAGACCGTCTACTGGGATCGAGACATGGAACAAAAGCTTATTTCTGAAGAAGACTTGTAATAGCTCGAG Amino Acid Sequence 1:SS-AGA2-K^(b)/β2m-c-myc (SEQ ID NO:23)MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTIEGRYPYDVPDYALQASGGGGSGGGGSGGGGSASGGLKGGPGGGSGGGGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGYYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEGGIGSGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEFTPTETDTYACRVKHDSMAEPKTVYWDRDMEQKLISEEDL**LE Nucleic Acid Sequence 2:SS-SIYR/K^(b)/β2m-c-myc-AGA2 (SEQ ID NO:24)GAATTCTACTTCATACATTTTCAATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTAGCAGCTAGCTTGGATAAAAGATCTATTTATAGATATTATGGTTTGCTTAAGGGTGGACCAGGTGGAGGTTCAGGAGGTGGAGGCCCACACTCGCTGAGGTATTTCGTCACCGCCGTGTCCCGGCCCGGCCTCGGGGAGCCCCGGTACATGGAAGTCGGCTACGTGGACGACACGGAGTTCGTGCGCTTCGACAGCGACGCGGAGAATCCGAGATATGAGCCGCGGGCGCGGTGGATGGAGCAGGAGGGGCCCGAGTATTGGGAGCGGGAGACACAGAAAGCCAAGGGCAATGAGCAGAGTTTCCGAGTGGACCTGAGGACCCTGCTCGGCTACTACAACCAGAGCAAGGGCGGCTCTCACACTATTCAGGTGATCTCTGGCTGTGAAGTGGGGTCCGACGGGCGACTCCTCCGCGGGTACCAGCAGTACGCCTACGACGGCTGCGATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACATGGCGGCGCTGATCACCAAACACAAGTGGGAGCAGGCTGGTGAAGCAGAGAGACTCAGGGCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCGCAGATACCTGAAGAACGGGAACGCGACGCTGCTGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTGAAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGACATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATGGAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGTGTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGGGTGGAATAGGTTCAGGTGGCGGTGGATCAGGAGGCGGAGGTTCAATCCAGAAAACCCCTCAAATTCAAGTATACTCACGCCACCCACCGGAGAATGGGAAGCCGAACATACTGAACTGCTACGTAACACAGTTCCACCCGCCTCACATTGAAATCCAAATGCTGAAGAACGGGAAAAAAATTCCTAAAGTAGAGATGTCAGATATGTCCTTCAGCAAGGACTGGTCTTTCTATATCCTGGCTCACACTGAATTCACCCCCACTGAGACTGATACATACGCCTGCAGAGTTAAGCATGACAGTATGGCCGAGCCCAAGACCGTCTACTGGGATCGAGACATGGAACAAAAGCTTATTTCTGAAGAAGACTTGCATATGCAGGAACTGACAACTATATGCGAGCAAATCCCCTCACCAACTTTAGAATCGACGCCGTACTCTTTGTCAACGACTACTATTTTGGCCAACGGGAAGGCAATGCAAGGAGTTTTTGAATATTACAAATCAGTAACGTTTGTCAGTAATTGCGGTTCTCACCCCTCAACAACTAGCAAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGGACAATAGCTCGACGATTGAAGGTAGATACCCATACGACGTTCCAGACTACGCTTAGTAACTCGAG Amino Acid Sequence 2:SS-SIYR/K^(b)/β2m-c-myc-AGA2 (SEQ ID NO:25)ILLHTFSIKMQLLRCFSIFSVIASVLAASLDKRSIYRYYGLLKGGPGGGSGGGGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGYYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEGGIGSGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEFTPTETDTYACRVKHDSMAEPKTVYWDRDMEQKLISEEDLHMQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTIEGRYPYDVPDYA Nucleic Acid Sequence 3:SS-dEV8/K^(b)/β2m-c-myc-AGA2 (SEQ ID NO:26)GAATTCTACTTCATACATTTTCAATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTAGCAGCTAGCTTGGATAAAAGAGAACAATACAAATTCTACTCAGTTCTTAAGGGTGGACCAGGTGGAGGTTCAGGAGGTGGAGGCCCACACTCGCTGAGGTATTTCGTCACCGCCGTGTCCCGGCCCGGCCTCGGGGAGCCCCGGTACATGGAAGTCGGCTACGTGGACGACACGGAGTTCGTGCGCTTCGACAGCGACGCGGAGAATCCGAGATATGAGCCGCGGGCGCGGTGGATGGAGCAGGAGGGGCCCGAGTATTGGGAGCGGGAGACACAGAAAGCCAAGGGCAATGAGCAGAGTTTCCGAGTGGACCTGAGGACCCTGCTCGGCTACTACAACCAGAGCAAGGGCGGCTCTCACACTATTCAGGTGATCTCTGGCTGTGAAGTGGGGTCCGACGGGCGACTCCTCCGCGGGTACCAGCAGTACGCCTACGACGGCTGCGATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACATGGCGGCGCTGATCACCAAACACAAGTGGGAGCAGGCTGGTGAAGCAGAGAGACTCAGGGCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCGCAGATACCTGAAGAACGGGAACGCGACGCTGCTGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTGAAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGACATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATGGAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGTGTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGGGTGGAATAGGTTCAGGTGGCGGTGGATCAGGAGGCGGAGGTTCAATCCAGAAAACCCCTCAAATTCAAGTATACTCACGCCACCCACCGGAGAATGGGAAGCCGAACATACTGAACTGCTACGTAACACAGTTCCACCCGCCTCACATTGAAATCCAAATGCTGAAGAAGAACGGAAAAATTCCTAAAGTAGAGATGTCAGATATGTCCTTCAGCAAGGACTGGTCTTTCTATATCCTGGCTCACACTGAATTCACCCCCACTGAGACTGATACATACGCCTGCAGAGTTAAGCATGACAGTATGGCCGAGCCCAAGACCGTCTACTGGGATCGAGACATGGAACAAAAGCTTATTTCTGAAGAAGACTTGCATATGCAGGAACTGACAACTATATGCGAGCAAATCCCCTCACCAACTTTAGAATCGACGCCGTACTCTTTGTCAACGACTACTATTTTGGCCAACGGGAAGGCAATGCAAGGAGTTTTTGAATATTACAAATCAGTAACGTTTGTCAGTAATTGCGGTTCTCACCCCTCAACAACTAGCAAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGGACAATAGCTCGACGATTGAAGGTAGATACCCATACGACGTTCCAGACTACGCTTAGTAACTCGAG Amino Acid Sequence 3:SS-dEV8/K^(b)/β2m-c-myc-AGA2 (SEQ ID NO:27)ILLHTFSIKMQLLRCFSIFSVIASVLAASLDKREQYKFYSVLKGGPGGGSGGGGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGYYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKEKLEGTCVEWLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFOKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEGGIGSGGGGSGGGGSIQKTPQIQWSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILATEFTPTETDTYACRVKHDSMAEPKTVYWDRDMEQKLISEEDLHQELTTICEQIPSPTLESTPYSLSTTTILANGKMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTIEGRYPYDVPDYA Nucleic Acid Sequence 4:SS-OVA/K^(b)/β2m-c-myc-AGA2 (SEQ ID NO:28)GAATTCTACTTCATACATTTTCAATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTAGCAGCTAGCTTGGATAAAAGGAGCATCATCAATTTTGAAAAGCTTCTTAAGGGTGGACCAGGTGGAGGTTCAGGAGGTGGAGGCCCACACTCGCTGAGGTATTTCGTCACCGCCGTGTCCCGGCCCGGCCTCGGGGAGCCCCGGTACATGGAAGTCGGCTACGTGGACGACACGGAGTTCGTGCGCTTCGACAGCGACGCGGAGAATCCGAGATATGAGCCGCGGGCGCGGTGGATGGAGCAGGAGGGGCCCGAGTATTGGGAGCGGGAGACACAGAAAGCCAAGGGCAATGAGCAGAGTTTCCGAGTGGACCTGAGGACCCTGCTCGGCTACTACAACCAGAGCAAGGGCGGCTCTCACACTATTCAGGTGATCTCTGGCTGTGAAGTGGGGTCCGACGGGCGACTCCTCCGCGGGTACCAGCAGTACGCCTACGACGGCTGCGATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACATGGCGGCGCTGATCACCAAACACAAGTGGGAGCAGGCTGGTGAAGCAGAGAGACTCAGGGCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCGCAGATACCTGAAGAACGGGAACGCGACGCTGCTGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTGAAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGACATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATGGAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGTGTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGGGTGGAATAGGTTCAGGTGGCGGTGGATCAGGAGGCGGAGGTTCAATCCAGAACCCCTCAARTTCAAGTATACTCACGCCACCCACCGGAGAATGGGAAGCCGAACATACTGAACTGCTACGTAACACAGTTCCACCCGCCTCACATTGAAATCCAAATGCTGAAGAACGGGAAAAAAATTCCTAAAGTAGAGATGTCAGATATGTCCTTCAGCAAGGACTGGTCTTTCTATATCCTGGCTCACACTGAATTCACCCCCACTGAGACTGATACATACGCCTGCAGAGTTAAGCATGACAGTATGGCCGAGCCCAAGACCGTCTACTGGGATCGAGACATGGAACAAAAGCTTATTTCTGAAGAAGACTTGCATATGCAGGAACTGACAACTATATGCGAGCAAATCCCCTCACCAACTTTAGAATCGACGCCGTACTCTTTGTCAACGACTACTATTTTGGCCAACGGGAGGCAATGCAAGGAAGAATATTACAAATCAGTAACGTTTGTCAGTAATTGCGGTTCTCACCCCTCAACAACTAGCAAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGGACAATAGCTCGACGATTGAAGGTAGATACCCATACGACGTTCCAGACTACGCTTAGTAACTCGAG Amino Acid Sequence 4:SS-OVA/K^(b)/β2m-c-myc-AGA2 (SEQ ID NO:29)ILLHTFSIKMQLLRCFSIFSVIASVLAASLDKRSIINFEKLLKGGPGGGSGGGGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGYYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEGGIGSGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEFTPETDTYACRVKHDSMAEPKTVYWDRDMEQKLISEEDLHMQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTIEGRYPYDVPDYA Nucleic Acid Sequence 5:SS-AGA2-L^(d)/β2m-c-myc (SEQ ID NO:30)ATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTAGCACAGGACTGACAACTATATGCGAGCAATCCCCTCACCTGGACGCCGTACTCTTTGTCAACGACTACTATTTTGGCCAACGGGAAGGCAATGCAAGGAGTTTTTGAATATTACAAATCAGTAACGTTTGTCAGTTTGCGGTTCTCACCCCTCAACAACTAGCAAAGGCAGCCCCATAAACACACAGTATGTTTTTGGACAATAGCTCGACGATTGAAGGTAGATACCCATACGACGTTCCAGACTACGCTCTGCAGGCTAGTGGTGGTGGTGGTTCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCGGTGGACTTAAGGGTGGACCAGGTGGAGGTTCAGGAGGTGGAGGCCCACACTCGATGCGGTATTTCGAGACCGCGGTGTCCCGGCGCGGCCTCGGGGAGCCCCGGTACATCTCTGTCGGCTATGTGAACGACAAGGAGTTCGTGCGCTTCGACAGCGACGCGGAGAATCCGAGATATGAGCCGAGGGCGCCGTGGATGGAGCAGGAGGGGCCGGAGTATTGGGAGCGGATCACGCAGATCGCCAAGGGCCAGGAGCAGTGGTTCCGAGTGAACCTGAGGACCCTGCTCGGCTACTACAACCAGAGCGCGGGCGGCACTCACACACTCCAGTGGATGTACGGCTGTGACGTGGGGTCGGACGGGCGCCTCCTCCGCGGGTACGAGCAGTTCGCCTACGACGGCTGCGATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGTTCGCGGACATGTCGTCGATGATCACCCGACGCAAGTGGGAGCAGGCTGGTGCTGCAGAGTATTACAGGGCCTACCTGGAGGGCGAGTGCGTGGAGTGGCTCCACAGATACCTGAAGAACGGGAATGCTACGCTGCTGCGCACAGATTCCCCAAAGGCACATGTGACCTATCACCCCAGATCTAAAGGTGAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGACATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGACCCAGGACATGGAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGAATTACACATGCCGTGTGTACCATGAGGGGCTGCCCCATCCCCTCACCCTGAGATGGGAGGGTGGAATAGGTTCAGGTGGCGGTGGATCAGGAGGCGGAGGTTCAATCCAGAAAACCCCTCAAATTCAAGTATACTCACGCCACCCACCGGAGAATGGGAAGCCGAACATACTGAACTGCTACGTAACACAGTTCCACCCGCCTCACATTGAAATCCAAATGCTGAAGAAGGGGAAAAAAATTCCTAAAGTAGAGATGTCAGATATGTCCTTCAGCAAGGACTGGTCTTTCTATATCCTGGCTCACACTGAATTCACCCCCACTGAGACTGATACATACGCCTGCAGAGTTAAGCATGACAGTATGGCCGAGCCCAAGACCGTCTACTGGGATCGAGACATGGAACAAAAGCTTATTTCTGAAGAAGAC TTGTAATAGCTCGAG AminoAcid Sequence 5: SS-AGA2-L^(d)/β2m-c-myc (SEQ ID NO:31)MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTIEGRYPYDVPDYALQASGGGGSGGGGSGGGGSASGGLKGGPGGGSGGGGPHSMRYFETAVSRRGLGEPRYISVGYVNDKEFVRFDSDAENPRYEPRAPWMEQEGPEYWERITQIAKGQEQWFRVNLRTLLGYYNQSAGGTHTLQWMYGCDVGSDGRLLRGYEQFAYDGCDYIALNEDLKTWTFADMSSMITRRKWEQAGAAEYYRAYLEGECVEWLHRYLKNGNATLLRTDSPKAHVTYHPRSKGEVTLRCWALGFYPADITLTWQLNGEELTQDMELVETRPAGDGTFQKWASVVVPLGKEQNYTCRVYHEGLPHPLTLRWEGGIGSGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEFTPTETDTYACRVKHDSMAEPKTVYWDRDMEQKLISEEDL

[0111] TABLE 3 Sequences of mutant clones isolated by sorting fromdEV8/Kb error-prone PCR library. Nucleic Acid Sequence 1: dEP.1 (SEQ IDNO:32) GAATTCTACTTCATACATTTTCAATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTAGCAGCTAGCTTGGATAAAAGAGAACAATACAAATTCTACTCAGTTCTTAAGGGTGGACCAGGTGGAGGTTCAGGAGGTGGAGGCCCACACTCGCTGAGGTATTTCGTCACCGCCGTGTCCCGGCCCGGCCTCGGGGAGCCCCGGTACATGGAAGTCGGCTACGTGGACGACACGGAGTTCGTGCGCTTCGACAGCGACGCGGAGAATCCGAGATATGAGCCGCGGGCGCGGTGGATGGAGCAGGAGGGGCCCGAGTATTGGGAGCGGGAGACACAGAAAGCCAAGGGCAATGAGCAGAGTTTCCGAGTGGACCTGAGGACCCTGCTCGGCTACTACAACCAGAGCAAGGGCGGCTCTCACACTATTCAGGTGATCTCTGGCTGTGGAATGGGGTCCGACGGGCGACTCCTCCGCGGGTACCAGCAGTACGCCTACGACGGCTGCGATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACATGGCGGCGCTGATCACCAAACACAAGTGGGAGCAGGCTGGTGAAGCAGAGAGACTCAGGGCCTACCTGGAGGGCACGTGCGTGGAGAGGCTCCGCAGATACCTGAAGAACGGGAACGCGACGCTGCTGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTGAAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGACATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCT Amino Acid Sequence 1: dEP.1 (SEQ IDNO:33) ILLHTFSIKMQLLRCFSIFSVIASVLAASLDKREQYKFYSVLKGGPGGGSGGGGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGYYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVERLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEGGIGSGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFNILAHTEFTPTETDTYACRVKHDSMAEPKTVYWDRDMEQRLISEEDLHMQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYESVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTIEGRYPYDVPDYA Nucleic Acid Sequence 2:dEP.3 (SEQ ID NO:34) GAATTCTACTTCATACATTTTCAATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTAGCAGCTAGCTTGGATAAAAGAGAACAATACAAATTCTACTCAGTTCTTAAGGGTGGACCAGGTGGAGGTTCAGGAGGTGGAGGCCCACACTCGCTGAGGTATTTCGTCACCGCCGTGTCCCGGCCCGGCCTCGGGGAGCCCCGGTACATGGAAGTCGGCTACGTGGACGACACGGAGTTCGTGCGCTTCGACAGCGACGCGGAGAATCCGAGATATGAGCCGCGGGCGCGGTGGATGGAGCAGGAGGGGCCCGAGTATTGGGAGCGGGAGACACAGAAAGCCAAGGGCAATGAGCAGAGTTTCCGAGTGGACCTGAGGACCCTGCTCGGCTACTACAACCAGAGCAAGGGCGGCTCTCACACTATTCAGGTGATCTCTGGCTGTGAAGTGGGGTCCGACGGGCGACTCCTCCGCGGGTACCAGCAGTACGCCTACGACGGCTGCGATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACATGGCGGCGCTGATCACCAAACACAAGTGGGAGCAGGCTGGTGAAGCAGAGAGACTCAGGGCCTACCTGGAGGGCACGTGCGTGGAGCGGCTCCGCAGATACCTGAAGAACGGGAACGCGACGCTGCT GCGCACAGATTCCCCAAAGGAmino Acid Sequence 2: dEP.3 (SEQ ID NO:35)ILLHTFSIKMQLLRCFSIFSVIASVLAASLDKREQYKFYSVLKGGPGGGSGGGGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGYYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVERLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEGGIGSGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEFTPTETDTYACRVKHDSMAEPKTVYWDRDMEQKLISEEDLHMQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTIEGRYPYDVPDYA Nucleic Acid Sequence 3:dEP.4 (SEQ ID NO:36) GAATTCTACTTCATACATTTTCAATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTAGCAGCTAGCTTGGATAAAAGAGAACAATACAAATTCTACTCAGTTCTTAAGGGTGGACCAGGTGGAGGTTCAGGAGGTGGAGGCCCACACTCGCTGAGGTATTTCGTCACCGCCGTGTCCCGGCCCGGCCTCGGGGAGCCCCGGTACATGGAAGTCGGCTACGTGGACGACACGGAGTTCGTGCGCTTCAACAGCGACGCGGAGAATCCGAGATATGAGCCGCGGGCGCGGTGGATGGAGCAGGAGGGGCCCGAGTATTGGGAGCGGGAGACACAGAAAGGCAAGGGCAATGAGCAGAGTTTCCGAGTGGACCTGAGGACCCTGCTCGGCTACTACAACCAGAGCAAGGGCGGCTCTCACACTATTCAGGTGATCTCTGGCTGTGAAGTGGGGTCCGACGGGCGACTCCTCCGCGGGTACCAGCAGTACGCCTACGACGGCTGCGATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACATGGCGGCGCTGATCACCAAACACAAGTGGGAGCAGGCTGGTGAAGCAGAGAGACTCAGGGCCTACCTGGAGGGCACGTGCGTGGAGAGGCTCCGCAGATACCTGAAGAACGGGAACGCGACGCTGCTGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTGAAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGACATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATGGAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGTGTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGGGTGGAATAGGTTCAGGTGGCGGTGGATCAGGAGGCGGAGGTTCAATCCAGAAAACCCCTCAAATTCAAGTATACTCACGCCACCCACCGGAGAATGGGAAGCCG Amino Acid Sequence 3: dEP.4(SEQ ID NO:37) ILLHTFSIKMQLLRCFSIFSVIASVLAASLDKREQYKFYSVLKGGPGGGSGGGGPHSLRYFVTAVSRPGLGEPRYMEVGVVDDTEFVRFNSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGYYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVERLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEGGIGSGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFNILAHTEFTPTETDTYACRVKHDSMAEPKTVYWDRDMEQRLISEEDLHMQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFKDNSSTIEGRYPYDVPDYA

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1 37 1 8 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 1 Ser Ile Tyr Arg Tyr Tyr Gly Leu 1 5 2 8 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide2 Ser Ile Ile Asn Phe Glu Lys Leu 1 5 3 63 DNA Artificial SequenceDescription of Artificial Sequence Synthetic nucleotide primer 3atattttctg ttattgcttc agttttagca gctagcttgg ataaaagann snnsnnsaaa 60 ttc63 4 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 4 Gln Leu Ser Pro Phe Pro Phe Asp Leu 1 5 5 8 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide5 Glu Gln Tyr Lys Phe Tyr Ser Val 1 5 6 8 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 6 Asn Asn Asn LysPhe Tyr Ser Val 1 5 7 79 DNA Artificial Sequence Description ofArtificial Sequence Synthetic nucleotide primer 7 caatggctag cggtggacttaagggtggac caggtggagg ttcaggaggt ggaggcccac 60 actcgctgag gtatttcgt 79 872 DNA Artificial Sequence Description of Artificial Sequence Syntheticnucleotide primer 8 tgaacctccg cctcctgatc caccgccacc tgaacctattccaccctccc atctcagggt 60 gaggggctca gg 72 9 73 DNA Artificial SequenceDescription of Artificial Sequence Synthetic nucleotide primer 9ggtggaatag gttcaggtgg cggtggatca ggaggcggag gttcaatcca gaaaacccct 60caaattcaag tat 73 10 71 DNA Artificial Sequence Description ofArtificial Sequence Synthetic nucleotide primer 10 gttccctcga gctattacaagtcttcttca gaaataagct tttgttccat gtctcgatcc 60 cagtagacgg t 71 11 27 DNAArtificial Sequence Description of Artificial Sequence Syntheticnucleotide primer 11 tctcaagaat tctacttcat acatttt 27 12 27 DNAArtificial Sequence Description of Artificial Sequence Syntheticnucleotide primer 12 gtatctgcta gctgctaaaa ctgaagc 27 13 72 DNAArtificial Sequence Description of Artificial Sequence Syntheticnucleotide primer 13 atactagcta gcttggataa aaggtctatt tatagatattatggtttgct taagggtgga 60 ccaggtggag gt 72 14 42 DNA Artificial SequenceDescription of Artificial Sequence Synthetic nucleotide primer 14caatccagat ctttactaat gcaagtcttc ttcagaaata ag 42 15 30 DNA ArtificialSequence Description of Artificial Sequence Synthetic nucleotide primer15 ggatatcata tgcaggaact gacaactata 30 16 43 DNA Artificial SequenceDescription of Artificial Sequence Synthetic nucleotide primer 16atttgcagat ctcgagttac taagcgtagt ctggaacgtc gta 43 17 42 DNA ArtificialSequence Description of Artificial Sequence Synthetic nucleotide primer17 ctagcttgga taaaaggagc atcatcaatt ttgaaaagct tc 42 18 42 DNAArtificial Sequence Description of Artificial Sequence Syntheticnucleotide primer 18 ttaagaagct tttcaaaatt gatgatgctc cttttatcca ag 4219 42 DNA Artificial Sequence Description of Artificial SequenceSynthetic nucleotide primer 19 ctagcttgga taaaagggaa caatacaaattctactcagt tc 42 20 42 DNA Artificial Sequence Description of ArtificialSequence Synthetic nucleotide primer 20 ttaagaactg agtagaattt gtattgttcccttttatcca ag 42 21 1632 DNA Artificial Sequence Description ofArtificial Sequence Synthetic nucleotide 21 atgcagttac ttcgctgtttttcaatattt tctgttattg cttcagtttt agcacaggaa 60 ctgacaacta tatgcgagcaaatcccctca ccaactttag aatcgacgcc gtactctttg 120 tcaacgacta ctattttggccaacgggaag gcaatgcaag gagtttttga atattacaaa 180 tcagtaacgt ttgtcagtaattgcggttct cacccctcaa caactagcaa aggcagcccc 240 ataaacacac agtatgtttttaaggacaat agctcgacga ttgaaggtag atacccatac 300 gacgttccag actacgctctgcaggctagt ggtggtggtg gttctggtgg tggtggttct 360 ggtggtggtg gttctgctagcggtggactt aagggtggac caggtggagg ttcaggaggt 420 ggaggcccac actcgctgaggtatttcgtc accgccgtgt cccggcccgg cctcggggag 480 ccccggtaca tggaagtcggctacgtggac gacacggagt tcgtgcgctt cgacagcgac 540 gcggagaatc cgagatatgagccgcgggcg cggtggatgg agcaggaggg gcccgagtat 600 tgggagcggg agacacagaaagccaagggc aatgagcaga gtttccgagt ggacctgagg 660 accctgctcg gctactacaaccagagcaag ggcggctctc acactattca ggtgatctct 720 ggctgtgaag tggggtccgacgggcgactc ctccgcgggt accagcagta cgcctacgac 780 ggctgcgatt acatcgccctgaacgaagac ctgaaaacgt ggacggcggc ggacatggcg 840 gcgctgatca ccaaacacaagtgggagcag gctggtgaag cagagagact cagggcctac 900 ctggagggca cgtgcgtggagtggctccgc agatacctga agaacgggaa cgcgacgctg 960 ctgcgcacag attccccaaaggcccatgtg acccatcaca gcagacctga agataaagtc 1020 accctgaggt gctgggccctgggcttctac cctgctgaca tcaccctgac ctggcagttg 1080 aatggggagg agctgatccaggacatggag cttgtggaga ccaggcctgc aggggatgga 1140 accttccaga agtgggcatctgtggtggtg cctcttggga aggagcagta ttacacatgc 1200 catgtgtacc atcaggggctgcctgagccc ctcaccctga gatgggaggg tggaataggt 1260 tcaggtggcg gtggatcaggaggcggaggt tcaatccaga aaacccctca aattcaagta 1320 tactcacgcc acccaccggagaatgggaag ccgaacatac tgaactgcta cgtaacacag 1380 ttccacccgc ctcacattgaaatccaaatg ctgaagaacg ggaaaaaaat tcctaaagta 1440 gagatgtcag atatgtccttcagcaaggac tggtctttct atatcctggc tcacactgaa 1500 ttcaccccca ctgagactgatacatacgcc tgcagagtta agcatgacag tatggccgag 1560 cccaagaccg tctactgggatcgagacatg gaacaaaagc ttatttctga agaagacttg 1620 taatagctcg ag 1632 22540 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 22 Met Gln Leu Leu Arg Cys Phe Ser Ile Phe Ser Val Ile Ala SerVal 1 5 10 15 Leu Ala Gln Glu Leu Thr Thr Ile Cys Glu Gln Ile Pro SerPro Thr 20 25 30 Leu Glu Ser Thr Pro Tyr Ser Leu Ser Thr Thr Thr Ile LeuAla Asn 35 40 45 Gly Lys Ala Met Gln Gly Val Phe Glu Tyr Tyr Lys Ser ValThr Phe 50 55 60 Val Ser Asn Cys Gly Ser His Pro Ser Thr Thr Ser Lys GlySer Pro 65 70 75 80 Ile Asn Thr Gln Tyr Val Phe Lys Asp Asn Ser Ser ThrIle Glu Gly 85 90 95 Arg Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Gln AlaSer Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly GlySer Ala Ser Gly 115 120 125 Gly Leu Lys Gly Gly Pro Gly Gly Gly Ser GlyGly Gly Gly Pro His 130 135 140 Ser Leu Arg Tyr Phe Val Thr Ala Val SerArg Pro Gly Leu Gly Glu 145 150 155 160 Pro Arg Tyr Met Glu Val Gly TyrVal Asp Asp Thr Glu Phe Val Arg 165 170 175 Phe Asp Ser Asp Ala Glu AsnPro Arg Tyr Glu Pro Arg Ala Arg Trp 180 185 190 Met Glu Gln Glu Gly ProGlu Tyr Trp Glu Arg Glu Thr Gln Lys Ala 195 200 205 Lys Gly Asn Glu GlnSer Phe Arg Val Asp Leu Arg Thr Leu Leu Gly 210 215 220 Tyr Tyr Asn GlnSer Lys Gly Gly Ser His Thr Ile Gln Val Ile Ser 225 230 235 240 Gly CysGlu Val Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr Gln Gln 245 250 255 TyrAla Tyr Asp Gly Cys Asp Tyr Ile Ala Leu Asn Glu Asp Leu Lys 260 265 270Thr Trp Thr Ala Ala Asp Met Ala Ala Leu Ile Thr Lys His Lys Trp 275 280285 Glu Gln Ala Gly Glu Ala Glu Arg Leu Arg Ala Tyr Leu Glu Gly Thr 290295 300 Cys Val Glu Trp Leu Arg Arg Tyr Leu Lys Asn Gly Asn Ala Thr Leu305 310 315 320 Leu Arg Thr Asp Ser Pro Lys Ala His Val Thr His His SerArg Pro 325 330 335 Glu Asp Lys Val Thr Leu Arg Cys Trp Ala Leu Gly PheTyr Pro Ala 340 345 350 Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu GluLeu Ile Gln Asp 355 360 365 Met Glu Leu Val Glu Thr Arg Pro Ala Gly AspGly Thr Phe Gln Lys 370 375 380 Trp Ala Ser Val Val Val Pro Leu Gly LysGlu Gln Tyr Tyr Thr Cys 385 390 395 400 His Val Tyr His Gln Gly Leu ProGlu Pro Leu Thr Leu Arg Trp Glu 405 410 415 Gly Gly Ile Gly Ser Gly GlyGly Gly Ser Gly Gly Gly Gly Ser Ile 420 425 430 Gln Lys Thr Pro Gln IleGln Val Tyr Ser Arg His Pro Pro Glu Asn 435 440 445 Gly Lys Pro Asn IleLeu Asn Cys Tyr Val Thr Gln Phe His Pro Pro 450 455 460 His Ile Glu IleGln Met Leu Lys Asn Gly Lys Lys Ile Pro Lys Val 465 470 475 480 Glu MetSer Asp Met Ser Phe Ser Lys Asp Trp Ser Phe Tyr Ile Leu 485 490 495 AlaHis Thr Glu Phe Thr Pro Thr Glu Thr Asp Thr Tyr Ala Cys Arg 500 505 510Val Lys His Asp Ser Met Ala Glu Pro Lys Thr Val Tyr Trp Asp Arg 515 520525 Asp Met Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 530 535 540 23 1640DNA Artificial Sequence Description of Artificial Sequence Syntheticnucleotide 23 gaattctact tcatacattt tcaattaaga tgcagttact tcgctgtttttcaatatttt 60 ctgttattgc ttcagtttta gcagctagct tggataaaag atctatttatagatattatg 120 gtttgcttaa gggtggacca ggtggaggtt caggaggtgg aggcccacactcgctgaggt 180 atttcgtcac cgccgtgtcc cggcccggcc tcggggagcc ccggtacatggaagtcggct 240 acgtggacga cacggagttc gtgcgcttcg acagcgacgc ggagaatccgagatatgagc 300 cgcgggcgcg gtggatggag caggaggggc ccgagtattg ggagcgggagacacagaaag 360 ccaagggcaa tgagcagagt ttccgagtgg acctgaggac cctgctcggctactacaacc 420 agagcaaggg cggctctcac actattcagg tgatctctgg ctgtgaagtggggtccgacg 480 ggcgactcct ccgcgggtac cagcagtacg cctacgacgg ctgcgattacatcgccctga 540 acgaagacct gaaaacgtgg acggcggcgg acatggcggc gctgatcaccaaacacaagt 600 gggagcaggc tggtgaagca gagagactca gggcctacct ggagggcacgtgcgtggagt 660 ggctccgcag atacctgaag aacgggaacg cgacgctgct gcgcacagattccccaaagg 720 cccatgtgac ccatcacagc agacctgaag ataaagtcac cctgaggtgctgggccctgg 780 gcttctaccc tgctgacatc accctgacct ggcagttgaa tggggaggagctgatccagg 840 acatggagct tgtggagacc aggcctgcag gggatggaac cttccagaagtgggcatctg 900 tggtggtgcc tcttgggaag gagcagtatt acacatgcca tgtgtaccatcaggggctgc 960 ctgagcccct caccctgaga tgggagggtg gaataggttc aggtggcggtggatcaggag 1020 gcggaggttc aatccagaaa acccctcaaa ttcaagtata ctcacgccacccaccggaga 1080 atgggaagcc gaacatactg aactgctacg taacacagtt ccacccgcctcacattgaaa 1140 tccaaatgct gaagaacggg aaaaaaattc ctaaagtaga gatgtcagatatgtccttca 1200 gcaaggactg gtctttctat atcctggctc acactgaatt cacccccactgagactgata 1260 catacgcctg cagagttaag catgacagta tggccgagcc caagaccgtctactgggatc 1320 gagacatgga acaaaagctt atttctgaag aagacttgca tatgcaggaactgacaacta 1380 tatgcgagca aatcccctca ccaactttag aatcgacgcc gtactctttgtcaacgacta 1440 ctattttggc caacgggaag gcaatgcaag gagtttttga atattacaaatcagtaacgt 1500 ttgtcagtaa ttgcggttct cacccctcaa caactagcaa aggcagccccataaacacac 1560 agtatgtttt taaggacaat agctcgacga ttgaaggtag atacccatacgacgttccag 1620 actacgctta gtaactcgag 1640 24 542 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 24 Ile LeuLeu His Thr Phe Ser Ile Lys Met Gln Leu Leu Arg Cys Phe 1 5 10 15 SerIle Phe Ser Val Ile Ala Ser Val Leu Ala Ala Ser Leu Asp Lys 20 25 30 ArgSer Ile Tyr Arg Tyr Tyr Gly Leu Leu Lys Gly Gly Pro Gly Gly 35 40 45 GlySer Gly Gly Gly Gly Pro His Ser Leu Arg Tyr Phe Val Thr Ala 50 55 60 ValSer Arg Pro Gly Leu Gly Glu Pro Arg Tyr Met Glu Val Gly Tyr 65 70 75 80Val Asp Asp Thr Glu Phe Val Arg Phe Asp Ser Asp Ala Glu Asn Pro 85 90 95Arg Tyr Glu Pro Arg Ala Arg Trp Met Glu Gln Glu Gly Pro Glu Tyr 100 105110 Trp Glu Arg Glu Thr Gln Lys Ala Lys Gly Asn Glu Gln Ser Phe Arg 115120 125 Val Asp Leu Arg Thr Leu Leu Gly Tyr Tyr Asn Gln Ser Lys Gly Gly130 135 140 Ser His Thr Ile Gln Val Ile Ser Gly Cys Glu Val Gly Ser AspGly 145 150 155 160 Arg Leu Leu Arg Gly Tyr Gln Gln Tyr Ala Tyr Asp GlyCys Asp Tyr 165 170 175 Ile Ala Leu Asn Glu Asp Leu Lys Thr Trp Thr AlaAla Asp Met Ala 180 185 190 Ala Leu Ile Thr Lys His Lys Trp Glu Gln AlaGly Glu Ala Glu Arg 195 200 205 Leu Arg Ala Tyr Leu Glu Gly Thr Cys ValGlu Trp Leu Arg Arg Tyr 210 215 220 Leu Lys Asn Gly Asn Ala Thr Leu LeuArg Thr Asp Ser Pro Lys Ala 225 230 235 240 His Val Thr His His Ser ArgPro Glu Asp Lys Val Thr Leu Arg Cys 245 250 255 Trp Ala Leu Gly Phe TyrPro Ala Asp Ile Thr Leu Thr Trp Gln Leu 260 265 270 Asn Gly Glu Glu LeuIle Gln Asp Met Glu Leu Val Glu Thr Arg Pro 275 280 285 Ala Gly Asp GlyThr Phe Gln Lys Trp Ala Ser Val Val Val Pro Leu 290 295 300 Gly Lys GluGln Tyr Tyr Thr Cys His Val Tyr His Gln Gly Leu Pro 305 310 315 320 GluPro Leu Thr Leu Arg Trp Glu Gly Gly Ile Gly Ser Gly Gly Gly 325 330 335Gly Ser Gly Gly Gly Gly Ser Ile Gln Lys Thr Pro Gln Ile Gln Val 340 345350 Tyr Ser Arg His Pro Pro Glu Asn Gly Lys Pro Asn Ile Leu Asn Cys 355360 365 Tyr Val Thr Gln Phe His Pro Pro His Ile Glu Ile Gln Met Leu Lys370 375 380 Asn Gly Lys Lys Ile Pro Lys Val Glu Met Ser Asp Met Ser PheSer 385 390 395 400 Lys Asp Trp Ser Phe Tyr Ile Leu Ala His Thr Glu PheThr Pro Thr 405 410 415 Glu Thr Asp Thr Tyr Ala Cys Arg Val Lys His AspSer Met Ala Glu 420 425 430 Pro Lys Thr Val Tyr Trp Asp Arg Asp Met GluGln Lys Leu Ile Ser 435 440 445 Glu Glu Asp Leu His Met Gln Glu Leu ThrThr Ile Cys Glu Gln Ile 450 455 460 Pro Ser Pro Thr Leu Glu Ser Thr ProTyr Ser Leu Ser Thr Thr Thr 465 470 475 480 Ile Leu Ala Asn Gly Lys AlaMet Gln Gly Val Phe Glu Tyr Tyr Lys 485 490 495 Ser Val Thr Phe Val SerAsn Cys Gly Ser His Pro Ser Thr Thr Ser 500 505 510 Lys Gly Ser Pro IleAsn Thr Gln Tyr Val Phe Lys Asp Asn Ser Ser 515 520 525 Thr Ile Glu GlyArg Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 530 535 540 25 1640 DNAArtificial Sequence Description of Artificial Sequence Syntheticnucleotide 25 gaattctact tcatacattt tcaattaaga tgcagttact tcgctgtttttcaatatttt 60 ctgttattgc ttcagtttta gcagctagct tggataaaag agaacaatacaaattctact 120 cagttcttaa gggtggacca ggtggaggtt caggaggtgg aggcccacactcgctgaggt 180 atttcgtcac cgccgtgtcc cggcccggcc tcggggagcc ccggtacatggaagtcggct 240 acgtggacga cacggagttc gtgcgcttcg acagcgacgc ggagaatccgagatatgagc 300 cgcgggcgcg gtggatggag caggaggggc ccgagtattg ggagcgggagacacagaaag 360 ccaagggcaa tgagcagagt ttccgagtgg acctgaggac cctgctcggctactacaacc 420 agagcaaggg cggctctcac actattcagg tgatctctgg ctgtgaagtggggtccgacg 480 ggcgactcct ccgcgggtac cagcagtacg cctacgacgg ctgcgattacatcgccctga 540 acgaagacct gaaaacgtgg acggcggcgg acatggcggc gctgatcaccaaacacaagt 600 gggagcaggc tggtgaagca gagagactca gggcctacct ggagggcacgtgcgtggagt 660 ggctccgcag atacctgaag aacgggaacg cgacgctgct gcgcacagattccccaaagg 720 cccatgtgac ccatcacagc agacctgaag ataaagtcac cctgaggtgctgggccctgg 780 gcttctaccc tgctgacatc accctgacct ggcagttgaa tggggaggagctgatccagg 840 acatggagct tgtggagacc aggcctgcag gggatggaac cttccagaagtgggcatctg 900 tggtggtgcc tcttgggaag gagcagtatt acacatgcca tgtgtaccatcaggggctgc 960 ctgagcccct caccctgaga tgggagggtg gaataggttc aggtggcggtggatcaggag 1020 gcggaggttc aatccagaaa acccctcaaa ttcaagtata ctcacgccacccaccggaga 1080 atgggaagcc gaacatactg aactgctacg taacacagtt ccacccgcctcacattgaaa 1140 tccaaatgct gaagaacggg aaaaaaattc ctaaagtaga gatgtcagatatgtccttca 1200 gcaaggactg gtctttctat atcctggctc acactgaatt cacccccactgagactgata 1260 catacgcctg cagagttaag catgacagta tggccgagcc caagaccgtctactgggatc 1320 gagacatgga acaaaagctt atttctgaag aagacttgca tatgcaggaactgacaacta 1380 tatgcgagca aatcccctca ccaactttag aatcgacgcc gtactctttgtcaacgacta 1440 ctattttggc caacgggaag gcaatgcaag gagtttttga atattacaaatcagtaacgt 1500 ttgtcagtaa ttgcggttct cacccctcaa caactagcaa aggcagccccataaacacac 1560 agtatgtttt taaggacaat agctcgacga ttgaaggtag atacccatacgacgttccag 1620 actacgctta gtaactcgag 1640 26 542 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 26 Ile LeuLeu His Thr Phe Ser Ile Lys Met Gln Leu Leu Arg Cys Phe 1 5 10 15 SerIle Phe Ser Val Ile Ala Ser Val Leu Ala Ala Ser Leu Asp Lys 20 25 30 ArgGlu Gln Tyr Lys Phe Tyr Ser Val Leu Lys Gly Gly Pro Gly Gly 35 40 45 GlySer Gly Gly Gly Gly Pro His Ser Leu Arg Tyr Phe Val Thr Ala 50 55 60 ValSer Arg Pro Gly Leu Gly Glu Pro Arg Tyr Met Glu Val Gly Tyr 65 70 75 80Val Asp Asp Thr Glu Phe Val Arg Phe Asp Ser Asp Ala Glu Asn Pro 85 90 95Arg Tyr Glu Pro Arg Ala Arg Trp Met Glu Gln Glu Gly Pro Glu Tyr 100 105110 Trp Glu Arg Glu Thr Gln Lys Ala Lys Gly Asn Glu Gln Ser Phe Arg 115120 125 Val Asp Leu Arg Thr Leu Leu Gly Tyr Tyr Asn Gln Ser Lys Gly Gly130 135 140 Ser His Thr Ile Gln Val Ile Ser Gly Cys Glu Val Gly Ser AspGly 145 150 155 160 Arg Leu Leu Arg Gly Tyr Gln Gln Tyr Ala Tyr Asp GlyCys Asp Tyr 165 170 175 Ile Ala Leu Asn Glu Asp Leu Lys Thr Trp Thr AlaAla Asp Met Ala 180 185 190 Ala Leu Ile Thr Lys His Lys Trp Glu Gln AlaGly Glu Ala Glu Arg 195 200 205 Leu Arg Ala Tyr Leu Glu Gly Thr Cys ValGlu Trp Leu Arg Arg Tyr 210 215 220 Leu Lys Asn Gly Asn Ala Thr Leu LeuArg Thr Asp Ser Pro Lys Ala 225 230 235 240 His Val Thr His His Ser ArgPro Glu Asp Lys Val Thr Leu Arg Cys 245 250 255 Trp Ala Leu Gly Phe TyrPro Ala Asp Ile Thr Leu Thr Trp Gln Leu 260 265 270 Asn Gly Glu Glu LeuIle Gln Asp Met Glu Leu Val Glu Thr Arg Pro 275 280 285 Ala Gly Asp GlyThr Phe Gln Lys Trp Ala Ser Val Val Val Pro Leu 290 295 300 Gly Lys GluGln Tyr Tyr Thr Cys His Val Tyr His Gln Gly Leu Pro 305 310 315 320 GluPro Leu Thr Leu Arg Trp Glu Gly Gly Ile Gly Ser Gly Gly Gly 325 330 335Gly Ser Gly Gly Gly Gly Ser Ile Gln Lys Thr Pro Gln Ile Gln Val 340 345350 Tyr Ser Arg His Pro Pro Glu Asn Gly Lys Pro Asn Ile Leu Asn Cys 355360 365 Tyr Val Thr Gln Phe His Pro Pro His Ile Glu Ile Gln Met Leu Lys370 375 380 Asn Gly Lys Lys Ile Pro Lys Val Glu Met Ser Asp Met Ser PheSer 385 390 395 400 Lys Asp Trp Ser Phe Tyr Ile Leu Ala His Thr Glu PheThr Pro Thr 405 410 415 Glu Thr Asp Thr Tyr Ala Cys Arg Val Lys His AspSer Met Ala Glu 420 425 430 Pro Lys Thr Val Tyr Trp Asp Arg Asp Met GluGln Lys Leu Ile Ser 435 440 445 Glu Glu Asp Leu His Met Gln Glu Leu ThrThr Ile Cys Glu Gln Ile 450 455 460 Pro Ser Pro Thr Leu Glu Ser Thr ProTyr Ser Leu Ser Thr Thr Thr 465 470 475 480 Ile Leu Ala Asn Gly Lys AlaMet Gln Gly Val Phe Glu Tyr Tyr Lys 485 490 495 Ser Val Thr Phe Val SerAsn Cys Gly Ser His Pro Ser Thr Thr Ser 500 505 510 Lys Gly Ser Pro IleAsn Thr Gln Tyr Val Phe Lys Asp Asn Ser Ser 515 520 525 Thr Ile Glu GlyArg Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 530 535 540 27 1640 DNAArtificial Sequence Description of Artificial Sequence Syntheticnucleotide 27 gaattctact tcatacattt tcaattaaga tgcagttact tcgctgtttttcaatatttt 60 ctgttattgc ttcagtttta gcagctagct tggataaaag gagcatcatcaattttgaaa 120 agcttcttaa gggtggacca ggtggaggtt caggaggtgg aggcccacactcgctgaggt 180 atttcgtcac cgccgtgtcc cggcccggcc tcggggagcc ccggtacatggaagtcggct 240 acgtggacga cacggagttc gtgcgcttcg acagcgacgc ggagaatccgagatatgagc 300 cgcgggcgcg gtggatggag caggaggggc ccgagtattg ggagcgggagacacagaaag 360 ccaagggcaa tgagcagagt ttccgagtgg acctgaggac cctgctcggctactacaacc 420 agagcaaggg cggctctcac actattcagg tgatctctgg ctgtgaagtggggtccgacg 480 ggcgactcct ccgcgggtac cagcagtacg cctacgacgg ctgcgattacatcgccctga 540 acgaagacct gaaaacgtgg acggcggcgg acatggcggc gctgatcaccaaacacaagt 600 gggagcaggc tggtgaagca gagagactca gggcctacct ggagggcacgtgcgtggagt 660 ggctccgcag atacctgaag aacgggaacg cgacgctgct gcgcacagattccccaaagg 720 cccatgtgac ccatcacagc agacctgaag ataaagtcac cctgaggtgctgggccctgg 780 gcttctaccc tgctgacatc accctgacct ggcagttgaa tggggaggagctgatccagg 840 acatggagct tgtggagacc aggcctgcag gggatggaac cttccagaagtgggcatctg 900 tggtggtgcc tcttgggaag gagcagtatt acacatgcca tgtgtaccatcaggggctgc 960 ctgagcccct caccctgaga tgggagggtg gaataggttc aggtggcggtggatcaggag 1020 gcggaggttc aatccagaaa acccctcaaa ttcaagtata ctcacgccacccaccggaga 1080 atgggaagcc gaacatactg aactgctacg taacacagtt ccacccgcctcacattgaaa 1140 tccaaatgct gaagaacggg aaaaaaattc ctaaagtaga gatgtcagatatgtccttca 1200 gcaaggactg gtctttctat atcctggctc acactgaatt cacccccactgagactgata 1260 catacgcctg cagagttaag catgacagta tggccgagcc caagaccgtctactgggatc 1320 gagacatgga acaaaagctt atttctgaag aagacttgca tatgcaggaactgacaacta 1380 tatgcgagca aatcccctca ccaactttag aatcgacgcc gtactctttgtcaacgacta 1440 ctattttggc caacgggaag gcaatgcaag gagtttttga atattacaaatcagtaacgt 1500 ttgtcagtaa ttgcggttct cacccctcaa caactagcaa aggcagccccataaacacac 1560 agtatgtttt taaggacaat agctcgacga ttgaaggtag atacccatacgacgttccag 1620 actacgctta gtaactcgag 1640 28 541 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 28 Ile LeuLeu His Thr Phe Ser Ile Lys Met Gln Leu Leu Arg Cys Phe 1 5 10 15 SerIle Phe Ser Val Ile Ala Ser Val Leu Ala Ala Ser Leu Asp Lys 20 25 30 ArgSer Ile Ile Asn Phe Glu Lys Leu Leu Lys Gly Gly Pro Gly Gly 35 40 45 GlySer Gly Gly Gly Gly Pro His Ser Leu Arg Tyr Phe Val Thr Ala 50 55 60 ValSer Arg Pro Gly Leu Gly Glu Pro Arg Tyr Met Glu Val Gly Tyr 65 70 75 80Val Asp Asp Thr Glu Phe Val Arg Phe Asp Ser Asp Ala Glu Asn Pro 85 90 95Arg Tyr Glu Pro Arg Ala Arg Trp Met Glu Gln Glu Gly Pro Glu Tyr 100 105110 Trp Glu Arg Glu Thr Gln Lys Ala Lys Gly Asn Glu Gln Ser Phe Arg 115120 125 Val Asp Leu Arg Thr Leu Leu Gly Tyr Tyr Asn Gln Ser Lys Gly Gly130 135 140 Ser His Thr Ile Gln Val Ile Ser Gly Cys Glu Val Gly Ser AspGly 145 150 155 160 Arg Leu Leu Arg Gly Tyr Gln Gln Tyr Ala Tyr Asp GlyCys Asp Tyr 165 170 175 Ile Ala Leu Asn Glu Asp Leu Lys Thr Trp Thr AlaAla Asp Met Ala 180 185 190 Ala Leu Ile Thr Lys His Lys Trp Glu Gln AlaGly Glu Ala Glu Arg 195 200 205 Leu Arg Ala Tyr Leu Glu Gly Thr Cys ValGlu Trp Leu Arg Arg Tyr 210 215 220 Leu Lys Asn Gly Asn Ala Thr Leu LeuArg Thr Asp Ser Pro Lys Ala 225 230 235 240 His Val Thr His His Ser ArgPro Glu Asp Lys Val Thr Leu Arg Cys 245 250 255 Trp Ala Leu Gly Phe TyrPro Ala Asp Ile Thr Leu Thr Trp Gln Leu 260 265 270 Asn Gly Glu Glu LeuIle Gln Asp Met Glu Leu Val Glu Thr Arg Pro 275 280 285 Ala Gly Asp GlyThr Phe Gln Lys Trp Ala Ser Val Val Val Pro Leu 290 295 300 Gly Lys GluGln Tyr Tyr Thr Cys His Val Tyr His Gln Gly Leu Pro 305 310 315 320 GluPro Leu Thr Leu Arg Trp Glu Gly Gly Ile Gly Ser Gly Gly Gly 325 330 335Gly Ser Gly Gly Gly Gly Ser Ile Gln Lys Thr Pro Gln Ile Gln Val 340 345350 Tyr Ser Arg His Pro Pro Glu Asn Gly Lys Pro Asn Ile Leu Asn Cys 355360 365 Tyr Val Thr Gln Phe His Pro Pro His Ile Glu Ile Gln Met Leu Lys370 375 380 Asn Gly Lys Lys Ile Pro Lys Val Glu Met Ser Asp Met Ser PheSer 385 390 395 400 Lys Asp Trp Ser Phe Tyr Ile Leu Ala His Thr Glu PheThr Pro Glu 405 410 415 Thr Asp Thr Tyr Ala Cys Arg Val Lys His Asp SerMet Ala Glu Pro 420 425 430 Lys Thr Val Tyr Trp Asp Arg Asp Met Glu GlnLys Leu Ile Ser Glu 435 440 445 Glu Asp Leu His Met Gln Glu Leu Thr ThrIle Cys Glu Gln Ile Pro 450 455 460 Ser Pro Thr Leu Glu Ser Thr Pro TyrSer Leu Ser Thr Thr Thr Ile 465 470 475 480 Leu Ala Asn Gly Lys Ala MetGln Gly Val Phe Glu Tyr Tyr Lys Ser 485 490 495 Val Thr Phe Val Ser AsnCys Gly Ser His Pro Ser Thr Thr Ser Lys 500 505 510 Gly Ser Pro Ile AsnThr Gln Tyr Val Phe Lys Asp Asn Ser Ser Thr 515 520 525 Ile Glu Gly ArgTyr Pro Tyr Asp Val Pro Asp Tyr Ala 530 535 540 29 1631 DNA ArtificialSequence Description of Artificial Sequence Synthetic nucleotide 29atgcagttac ttcgctgttt ttcaatattt tctgttattg cttcagtttt agcacaggaa 60ctgacaacta tatgcgagca aatcccctca ccaactttag aatcgacgcc gtactctttg 120tcaacgacta ctattttggc caacgggaag gcaatgcaag gagtttttga atattacaaa 180tcagtaacgt ttgtcagtaa ttgcggttct cacccctcaa caactagcaa aggcagcccc 240ataaacacac agtatgtttt taaggacaat agctcgacga ttgaaggtag atacccatac 300gacgttccag actacgctct gcaggctagt ggtggtggtg gttcggtggt ggtggttctg 360gtggtggtgg ttctgctagc ggtggactta agggtggacc aggtggaggt tcaggaggtg 420gaggcccaca ctcgatgcgg tatttcgaga ccgcggtgtc ccggcgcggc ctcggggagc 480cccggtacat ctctgtcggc tatgtgaacg acaaggagtt cgtgcgcttc gacagcgacg 540cggagaatcc gagatatgag ccgagggcgc cgtggatgga gcaggagggg ccggagtatt 600gggagcggat cacgcagatc gccaagggcc aggagcagtg gttccgagtg aacctgagga 660ccctgctcgg ctactacaac cagagcgcgg gcggcactca cacactccag tggatgtacg 720gctgtgacgt ggggtcggac gggcgcctcc tccgcgggta cgagcagttc gcctacgacg 780gctgcgatta catcgccctg aacgaagacc tgaaaacgtg gacgttcgcg gacatgtcgt 840cgatgatcac ccgacgcaag tgggagcagg ctggtgctgc agagtattac agggcctacc 900tggagggcga gtgcgtggag tggctccaca gatacctgaa gaacgggaat gctacgctgc 960tgcgcacaga ttccccaaag gcacatgtga cctatcaccc cagatctaaa ggtgaagtca 1020ccctgaggtg ctgggccctg ggcttctacc ctgctgacat caccctgacc tggcagttga 1080atggggagga gctgacccag gacatggagc ttgtggagac caggcctgca ggggatggaa 1140ccttccagaa gtgggcatct gtggtggtgc ctcttgggaa ggagcagaat tacacatgcc 1200gtgtgtacca tgaggggctg ccccatcccc tcaccctgag atgggagggt ggaataggtt 1260caggtggcgg tggatcagga ggcggaggtt caatccagaa aacccctcaa attcaagtat 1320actcacgcca cccaccggag aatgggaagc cgaacatact gaactgctac gtaacacagt 1380tccacccgcc tcacattgaa atccaaatgc tgaagaacgg gaaaaaaatt cctaaagtag 1440agatgtcaga tatgtccttc agcaaggact ggtctttcta tatcctggct cacactgaat 1500tcacccccac tgagactgat acatacgcct gcagagttaa gcatgacagt atggccgagc 1560ccaagaccgt ctactgggat cgagacatgg aacaaaagct tatttctgaa gaagacttgt 1620aatagctcga g 1631 30 540 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 30 Met Gln Leu Leu Arg Cys Phe SerIle Phe Ser Val Ile Ala Ser Val 1 5 10 15 Leu Ala Gln Glu Leu Thr ThrIle Cys Glu Gln Ile Pro Ser Pro Thr 20 25 30 Leu Glu Ser Thr Pro Tyr SerLeu Ser Thr Thr Thr Ile Leu Ala Asn 35 40 45 Gly Lys Ala Met Gln Gly ValPhe Glu Tyr Tyr Lys Ser Val Thr Phe 50 55 60 Val Ser Asn Cys Gly Ser HisPro Ser Thr Thr Ser Lys Gly Ser Pro 65 70 75 80 Ile Asn Thr Gln Tyr ValPhe Lys Asp Asn Ser Ser Thr Ile Glu Gly 85 90 95 Arg Tyr Pro Tyr Asp ValPro Asp Tyr Ala Leu Gln Ala Ser Gly Gly 100 105 110 Gly Gly Ser Gly GlyGly Gly Ser Gly Gly Gly Gly Ser Ala Ser Gly 115 120 125 Gly Leu Lys GlyGly Pro Gly Gly Gly Ser Gly Gly Gly Gly Pro His 130 135 140 Ser Met ArgTyr Phe Glu Thr Ala Val Ser Arg Arg Gly Leu Gly Glu 145 150 155 160 ProArg Tyr Ile Ser Val Gly Tyr Val Asn Asp Lys Glu Phe Val Arg 165 170 175Phe Asp Ser Asp Ala Glu Asn Pro Arg Tyr Glu Pro Arg Ala Pro Trp 180 185190 Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Arg Ile Thr Gln Ile Ala 195200 205 Lys Gly Gln Glu Gln Trp Phe Arg Val Asn Leu Arg Thr Leu Leu Gly210 215 220 Tyr Tyr Asn Gln Ser Ala Gly Gly Thr His Thr Leu Gln Trp MetTyr 225 230 235 240 Gly Cys Asp Val Gly Ser Asp Gly Arg Leu Leu Arg GlyTyr Glu Gln 245 250 255 Phe Ala Tyr Asp Gly Cys Asp Tyr Ile Ala Leu AsnGlu Asp Leu Lys 260 265 270 Thr Trp Thr Phe Ala Asp Met Ser Ser Met IleThr Arg Arg Lys Trp 275 280 285 Glu Gln Ala Gly Ala Ala Glu Tyr Tyr ArgAla Tyr Leu Glu Gly Glu 290 295 300 Cys Val Glu Trp Leu His Arg Tyr LeuLys Asn Gly Asn Ala Thr Leu 305 310 315 320 Leu Arg Thr Asp Ser Pro LysAla His Val Thr Tyr His Pro Arg Ser 325 330 335 Lys Gly Glu Val Thr LeuArg Cys Trp Ala Leu Gly Phe Tyr Pro Ala 340 345 350 Asp Ile Thr Leu ThrTrp Gln Leu Asn Gly Glu Glu Leu Thr Gln Asp 355 360 365 Met Glu Leu ValGlu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys 370 375 380 Trp Ala SerVal Val Val Pro Leu Gly Lys Glu Gln Asn Tyr Thr Cys 385 390 395 400 ArgVal Tyr His Glu Gly Leu Pro His Pro Leu Thr Leu Arg Trp Glu 405 410 415Gly Gly Ile Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile 420 425430 Gln Lys Thr Pro Gln Ile Gln Val Tyr Ser Arg His Pro Pro Glu Asn 435440 445 Gly Lys Pro Asn Ile Leu Asn Cys Tyr Val Thr Gln Phe His Pro Pro450 455 460 His Ile Glu Ile Gln Met Leu Lys Asn Gly Lys Lys Ile Pro LysVal 465 470 475 480 Glu Met Ser Asp Met Ser Phe Ser Lys Asp Trp Ser PheTyr Ile Leu 485 490 495 Ala His Thr Glu Phe Thr Pro Thr Glu Thr Asp ThrTyr Ala Cys Arg 500 505 510 Val Lys His Asp Ser Met Ala Glu Pro Lys ThrVal Tyr Trp Asp Arg 515 520 525 Asp Met Glu Gln Lys Leu Ile Ser Glu GluAsp Leu 530 535 540 31 832 DNA Artificial Sequence Description ofArtificial Sequence Synthetic nucleotide 31 gaattctact tcatacattttcaattaaga tgcagttact tcgctgtttt tcaatatttt 60 ctgttattgc ttcagttttagcagctagct tggataaaag agaacaatac aaattctact 120 cagttcttaa gggtggaccaggtggaggtt caggaggtgg aggcccacac tcgctgaggt 180 atttcgtcac cgccgtgtcccggcccggcc tcggggagcc ccggtacatg gaagtcggct 240 acgtggacga cacggagttcgtgcgcttcg acagcgacgc ggagaatccg agatatgagc 300 cgcgggcgcg gtggatggagcaggaggggc ccgagtattg ggagcgggag acacagaaag 360 ccaagggcaa tgagcagagtttccgagtgg acctgaggac cctgctcggc tactacaacc 420 agagcaaggg cggctctcacactattcagg tgatctctgg ctgtgaagtg gggtccgacg 480 ggcgactcct ccgcgggtaccagcagtacg cctacgacgg ctgcgattac atcgccctga 540 acgaagacct gaaaacgtggacggcggcgg acatggcggc gctgatcacc aaacacaagt 600 gggagcaggc tggtgaagcagagagactca gggcctacct ggagggcacg tgcgtggaga 660 ggctccgcag atacctgaagaacgggaacg cgacgctgct gcgcacagat tccccaaagg 720 cccatgtgac ccatcacagcagacctgaag ataaagtcac cctgaggtgc tgggccctgg 780 gcttctaccc tgctgacatcaccctgacct ggcagttgaa tggggaggag ct 832 32 542 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 32 Ile Leu Leu HisThr Phe Ser Ile Lys Met Gln Leu Leu Arg Cys Phe 1 5 10 15 Ser Ile PheSer Val Ile Ala Ser Val Leu Ala Ala Ser Leu Asp Lys 20 25 30 Arg Glu GlnTyr Lys Phe Tyr Ser Val Leu Lys Gly Gly Pro Gly Gly 35 40 45 Gly Ser GlyGly Gly Gly Pro His Ser Leu Arg Tyr Phe Val Thr Ala 50 55 60 Val Ser ArgPro Gly Leu Gly Glu Pro Arg Tyr Met Glu Val Gly Tyr 65 70 75 80 Val AspAsp Thr Glu Phe Val Arg Phe Asp Ser Asp Ala Glu Asn Pro 85 90 95 Arg TyrGlu Pro Arg Ala Arg Trp Met Glu Gln Glu Gly Pro Glu Tyr 100 105 110 TrpGlu Arg Glu Thr Gln Lys Ala Lys Gly Asn Glu Gln Ser Phe Arg 115 120 125Val Asp Leu Arg Thr Leu Leu Gly Tyr Tyr Asn Gln Ser Lys Gly Gly 130 135140 Ser His Thr Ile Gln Val Ile Ser Gly Cys Glu Val Gly Ser Asp Gly 145150 155 160 Arg Leu Leu Arg Gly Tyr Gln Gln Tyr Ala Tyr Asp Gly Cys AspTyr 165 170 175 Ile Ala Leu Asn Glu Asp Leu Lys Thr Trp Thr Ala Ala AspMet Ala 180 185 190 Ala Leu Ile Thr Lys His Lys Trp Glu Gln Ala Gly GluAla Glu Arg 195 200 205 Leu Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu ArgLeu Arg Arg Tyr 210 215 220 Leu Lys Asn Gly Asn Ala Thr Leu Leu Arg ThrAsp Ser Pro Lys Ala 225 230 235 240 His Val Thr His His Ser Arg Pro GluAsp Lys Val Thr Leu Arg Cys 245 250 255 Trp Ala Leu Gly Phe Tyr Pro AlaAsp Ile Thr Leu Thr Trp Gln Leu 260 265 270 Asn Gly Glu Glu Leu Ile GlnAsp Met Glu Leu Val Glu Thr Arg Pro 275 280 285 Ala Gly Asp Gly Thr PheGln Lys Trp Ala Ser Val Val Val Pro Leu 290 295 300 Gly Lys Glu Gln TyrTyr Thr Cys His Val Tyr His Gln Gly Leu Pro 305 310 315 320 Glu Pro LeuThr Leu Arg Trp Glu Gly Gly Ile Gly Ser Gly Gly Gly 325 330 335 Gly SerGly Gly Gly Gly Ser Ile Gln Lys Thr Pro Gln Ile Gln Val 340 345 350 TyrSer Arg His Pro Pro Glu Asn Gly Lys Pro Asn Ile Leu Asn Cys 355 360 365Tyr Val Thr Gln Phe His Pro Pro His Ile Glu Ile Gln Met Leu Lys 370 375380 Asn Gly Lys Lys Ile Pro Lys Val Glu Met Ser Asp Met Ser Phe Ser 385390 395 400 Lys Asp Trp Ser Phe Asn Ile Leu Ala His Thr Glu Phe Thr ProThr 405 410 415 Glu Thr Asp Thr Tyr Ala Cys Arg Val Lys His Asp Ser MetAla Glu 420 425 430 Pro Lys Thr Val Tyr Trp Asp Arg Asp Met Glu Gln ArgLeu Ile Ser 435 440 445 Glu Glu Asp Leu His Met Gln Glu Leu Thr Thr IleCys Glu Gln Ile 450 455 460 Pro Ser Pro Thr Leu Glu Ser Thr Pro Tyr SerLeu Ser Thr Thr Thr 465 470 475 480 Ile Leu Ala Asn Gly Lys Ala Met GlnGly Val Phe Glu Tyr Tyr Lys 485 490 495 Ser Val Thr Phe Val Ser Asn CysGly Ser His Pro Ser Thr Thr Ser 500 505 510 Lys Gly Ser Pro Ile Asn ThrGln Tyr Val Phe Lys Asp Asn Ser Ser 515 520 525 Thr Ile Glu Gly Arg TyrPro Tyr Asp Val Pro Asp Tyr Ala 530 535 540 33 720 DNA ArtificialSequence Description of Artificial Sequence Synthetic nucleotide 33gaattctact tcatacattt tcaattaaga tgcagttact tcgctgtttt tcaatatttt 60ctgttattgc ttcagtttta gcagctagct tggataaaag agaacaatac aaattctact 120cagttcttaa gggtggacca ggtggaggtt caggaggtgg aggcccacac tcgctgaggt 180atttcgtcac cgccgtgtcc cggcccggcc tcggggagcc ccggtacatg gaagtcggct 240acgtggacga cacggagttc gtgcgcttcg acagcgacgc ggagaatccg agatatgagc 300cgcgggcgcg gtggatggag caggaggggc ccgagtattg ggagcgggag acacagaaag 360ccaagggcaa tgagcagagt ttccgagtgg acctgaggac cctgctcggc tactacaacc 420agagcaaggg cggctctcac actattcagg tgatctctgg ctgtgaagtg gggtccgacg 480ggcgactcct ccgcgggtac cagcagtacg cctacgacgg ctgcgattac atcgccctga 540acgaagacct gaaaacgtgg acggcggcgg acatggcggc gctgatcacc aaacacaagt 600gggagcaggc tggtgaagca gagagactca gggcctacct ggagggcacg tgcgtggagc 660ggctccgcag atacctgaag aacgggaacg cgacgctgct gcgcacagat tccccaaagg 720 34542 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 34 Ile Leu Leu His Thr Phe Ser Ile Lys Met Gln Leu Leu Arg CysPhe 1 5 10 15 Ser Ile Phe Ser Val Ile Ala Ser Val Leu Ala Ala Ser LeuAsp Lys 20 25 30 Arg Glu Gln Tyr Lys Phe Tyr Ser Val Leu Lys Gly Gly ProGly Gly 35 40 45 Gly Ser Gly Gly Gly Gly Pro His Ser Leu Arg Tyr Phe ValThr Ala 50 55 60 Val Ser Arg Pro Gly Leu Gly Glu Pro Arg Tyr Met Glu ValGly Tyr 65 70 75 80 Val Asp Asp Thr Glu Phe Val Arg Phe Asp Ser Asp AlaGlu Asn Pro 85 90 95 Arg Tyr Glu Pro Arg Ala Arg Trp Met Glu Gln Glu GlyPro Glu Tyr 100 105 110 Trp Glu Arg Glu Thr Gln Lys Ala Lys Gly Asn GluGln Ser Phe Arg 115 120 125 Val Asp Leu Arg Thr Leu Leu Gly Tyr Tyr AsnGln Ser Lys Gly Gly 130 135 140 Ser His Thr Ile Gln Val Ile Ser Gly CysGlu Val Gly Ser Asp Gly 145 150 155 160 Arg Leu Leu Arg Gly Tyr Gln GlnTyr Ala Tyr Asp Gly Cys Asp Tyr 165 170 175 Ile Ala Leu Asn Glu Asp LeuLys Thr Trp Thr Ala Ala Asp Met Ala 180 185 190 Ala Leu Ile Thr Lys HisLys Trp Glu Gln Ala Gly Glu Ala Glu Arg 195 200 205 Leu Arg Ala Tyr LeuGlu Gly Thr Cys Val Glu Arg Leu Arg Arg Tyr 210 215 220 Leu Lys Asn GlyAsn Ala Thr Leu Leu Arg Thr Asp Ser Pro Lys Ala 225 230 235 240 His ValThr His His Ser Arg Pro Glu Asp Lys Val Thr Leu Arg Cys 245 250 255 TrpAla Leu Gly Phe Tyr Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu 260 265 270Asn Gly Glu Glu Leu Ile Gln Asp Met Glu Leu Val Glu Thr Arg Pro 275 280285 Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ser Val Val Val Pro Leu 290295 300 Gly Lys Glu Gln Tyr Tyr Thr Cys His Val Tyr His Gln Gly Leu Pro305 310 315 320 Glu Pro Leu Thr Leu Arg Trp Glu Gly Gly Ile Gly Ser GlyGly Gly 325 330 335 Gly Ser Gly Gly Gly Gly Ser Ile Gln Lys Thr Pro GlnIle Gln Val 340 345 350 Tyr Ser Arg His Pro Pro Glu Asn Gly Lys Pro AsnIle Leu Asn Cys 355 360 365 Tyr Val Thr Gln Phe His Pro Pro His Ile GluIle Gln Met Leu Lys 370 375 380 Asn Gly Lys Lys Ile Pro Lys Val Glu MetSer Asp Met Ser Phe Ser 385 390 395 400 Lys Asp Trp Ser Phe Tyr Ile LeuAla His Thr Glu Phe Thr Pro Thr 405 410 415 Glu Thr Asp Thr Tyr Ala CysArg Val Lys His Asp Ser Met Ala Glu 420 425 430 Pro Lys Thr Val Tyr TrpAsp Arg Asp Met Glu Gln Lys Leu Ile Ser 435 440 445 Glu Glu Asp Leu HisMet Gln Glu Leu Thr Thr Ile Cys Glu Gln Ile 450 455 460 Pro Ser Pro ThrLeu Glu Ser Thr Pro Tyr Ser Leu Ser Thr Thr Thr 465 470 475 480 Ile LeuAla Asn Gly Lys Ala Met Gln Gly Val Phe Glu Tyr Tyr Lys 485 490 495 SerVal Thr Phe Val Ser Asn Cys Gly Ser His Pro Ser Thr Thr Ser 500 505 510Lys Gly Ser Pro Ile Asn Thr Gln Tyr Val Phe Lys Asp Asn Ser Ser 515 520525 Thr Ile Glu Gly Arg Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 530 535 54035 1091 DNA Artificial Sequence Description of Artificial SequenceSynthetic nucleotide 35 gaattctact tcatacattt tcaattaaga tgcagttacttcgctgtttt tcaatatttt 60 ctgttattgc ttcagtttta gcagctagct tggataaaagagaacaatac aaattctact 120 cagttcttaa gggtggacca ggtggaggtt caggaggtggaggcccacac tcgctgaggt 180 atttcgtcac cgccgtgtcc cggcccggcc tcggggagccccggtacatg gaagtcggct 240 acgtggacga cacggagttc gtgcgcttca acagcgacgcggagaatccg agatatgagc 300 cgcgggcgcg gtggatggag caggaggggc ccgagtattgggagcgggag acacagaaag 360 ccaagggcaa tgagcagagt ttccgagtgg acctgaggaccctgctcggc tactacaacc 420 agagcaaggg cggctctcac actattcagg tgatctctggctgtgaagtg gggtccgacg 480 ggcgactcct ccgcgggtac cagcagtacg cctacgacggctgcgattac atcgccctga 540 acgaagacct gaaaacgtgg acggcggcgg acatggcggcgctgatcacc aaacacaagt 600 gggagcaggc tggtgaagca gagagactca gggcctacctggagggcacg tgcgtggaga 660 ggctccgcag atacctgaag aacgggaacg cgacgctgctgcgcacagat tccccaaagg 720 cccatgtgac ccatcacagc agacctgaag ataaagtcaccctgaggtgc tgggccctgg 780 gcttctaccc tgctgacatc accctgacct ggcagttgaatggggaggag ctgatccagg 840 acatggagct tgtggagacc aggcctgcag gggatggaaccttccagaag tgggcatctg 900 tggtggtgcc tcttgggaag gagcagtatt acacatgccatgtgtaccat caggggctgc 960 ctgagcccct caccctgaga tgggagggtg gaataggttcaggtggcggt ggatcaggag 1020 gcggaggttc aatccagaaa acccctcaaa ttcaagtatactcacgccac ccaccggaga 1080 atgggaagcc g 1091 36 542 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 36 Ile LeuLeu His Thr Phe Ser Ile Lys Met Gln Leu Leu Arg Cys Phe 1 5 10 15 SerIle Phe Ser Val Ile Ala Ser Val Leu Ala Ala Ser Leu Asp Lys 20 25 30 ArgGlu Gln Tyr Lys Phe Tyr Ser Val Leu Lys Gly Gly Pro Gly Gly 35 40 45 GlySer Gly Gly Gly Gly Pro His Ser Leu Arg Tyr Phe Val Thr Ala 50 55 60 ValSer Arg Pro Gly Leu Gly Glu Pro Arg Tyr Met Glu Val Gly Tyr 65 70 75 80Val Asp Asp Thr Glu Phe Val Arg Phe Asn Ser Asp Ala Glu Asn Pro 85 90 95Arg Tyr Glu Pro Arg Ala Arg Trp Met Glu Gln Glu Gly Pro Glu Tyr 100 105110 Trp Glu Arg Glu Thr Gln Lys Ala Lys Gly Asn Glu Gln Ser Phe Arg 115120 125 Val Asp Leu Arg Thr Leu Leu Gly Tyr Tyr Asn Gln Ser Lys Gly Gly130 135 140 Ser His Thr Ile Gln Val Ile Ser Gly Cys Glu Val Gly Ser AspGly 145 150 155 160 Arg Leu Leu Arg Gly Tyr Gln Gln Tyr Ala Tyr Asp GlyCys Asp Tyr 165 170 175 Ile Ala Leu Asn Glu Asp Leu Lys Thr Trp Thr AlaAla Asp Met Ala 180 185 190 Ala Leu Ile Thr Lys His Lys Trp Glu Gln AlaGly Glu Ala Glu Arg 195 200 205 Leu Arg Ala Tyr Leu Glu Gly Thr Cys ValGlu Arg Leu Arg Arg Tyr 210 215 220 Leu Lys Asn Gly Asn Ala Thr Leu LeuArg Thr Asp Ser Pro Lys Ala 225 230 235 240 His Val Thr His His Ser ArgPro Glu Asp Lys Val Thr Leu Arg Cys 245 250 255 Trp Ala Leu Gly Phe TyrPro Ala Asp Ile Thr Leu Thr Trp Gln Leu 260 265 270 Asn Gly Glu Glu LeuIle Gln Asp Met Glu Leu Val Glu Thr Arg Pro 275 280 285 Ala Gly Asp GlyThr Phe Gln Lys Trp Ala Ser Val Val Val Pro Leu 290 295 300 Gly Lys GluGln Tyr Tyr Thr Cys His Val Tyr His Gln Gly Leu Pro 305 310 315 320 GluPro Leu Thr Leu Arg Trp Glu Gly Gly Ile Gly Ser Gly Gly Gly 325 330 335Gly Ser Gly Gly Gly Gly Ser Ile Gln Lys Thr Pro Gln Ile Gln Val 340 345350 Tyr Ser Arg His Pro Pro Glu Asn Gly Lys Pro Asn Ile Leu Asn Cys 355360 365 Tyr Val Thr Gln Phe His Pro Pro His Ile Glu Ile Gln Met Leu Lys370 375 380 Asn Gly Lys Lys Ile Pro Lys Val Glu Met Ser Asp Met Ser PheSer 385 390 395 400 Lys Asp Trp Ser Phe Asn Ile Leu Ala His Thr Glu PheThr Pro Thr 405 410 415 Glu Thr Asp Thr Tyr Ala Cys Arg Val Lys His AspSer Met Ala Glu 420 425 430 Pro Lys Thr Val Tyr Trp Asp Arg Asp Met GluGln Arg Leu Ile Ser 435 440 445 Glu Glu Asp Leu His Met Gln Glu Leu ThrThr Ile Cys Glu Gln Ile 450 455 460 Pro Ser Pro Thr Leu Glu Ser Thr ProTyr Ser Leu Ser Thr Thr Thr 465 470 475 480 Ile Leu Ala Asn Gly Lys AlaMet Gln Gly Val Phe Glu Tyr Tyr Lys 485 490 495 Ser Val Thr Phe Val SerAsn Cys Gly Ser His Pro Ser Thr Thr Ser 500 505 510 Lys Gly Ser Pro IleAsn Thr Gln Tyr Val Phe Lys Asp Asn Ser Ser 515 520 525 Thr Ile Glu GlyArg Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 530 535 540 37 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 37 His TyrSer Pro Phe Arg Gln Leu Ala 1 5

What is claimed is:
 1. A mutagenized combinatorial library of MajorHistocompatibility Complex (MHC) Class I chimeric proteins displayed onthe surfaces of recombinant yeast cells, wherein the mutagenizedcombinatorial library comprises at least one member MHC Class I chimericprotein which is improved in conformational stability or in specifictarget binding as compared with a comparison MHC Class I chimericprotein which has not been mutagenized.
 2. The mutagenized combinatoriallibrary of claim 1 wherein the MHC Class I chimeric protein comprises aportion mediating binding to the surfaces of the recombinant yeast cellsand a portion which comprises a specific target binding region of a MHCClass I protein.
 3. The mutagenized combinatorial library of claim 2wherein the portion mediated binding to the surfaces of the recombinantyeast cells is a mating adhesion receptor portion.
 4. The mutagenizedcombinatorial library of claim 3 wherein the mating adhesion receptorportion is an AGA2 portion.
 5. The mutagenized combinatorial library ofany of claims 2 to 4 wherein the chimeric protein further comprises aportion characterized by an amino acid sequence of a peptide which bindsto the binding region of the MHC Class I chimeric protein.
 6. Themutagenized combinatorial library of any of claims 2 to 5 wherein thechimeric protein further comprises a portion derived from a c-mycprotein and which mediates binding to a c-myc specific antibody.
 7. Themutagenized combinatorial library of any of claims 2 to 6 wherein thebinding region of the MHC Class I chimeric protein specifically binds aspecific target selected from the group consisting of a neoplastic cell,a virus-infected cell, a fungus-infected cell, a parasite-infected celland a bacterium-infected cell.
 8. The mutagenized combinatorial libraryof claim 8 wherein the peptide binding region specifically binds apeptide having the amino acid sequence given in SEQ ID NO:19, SEQ IDNO:22 or SEQ ID NO:24.
 9. The mutagenized combinatorial library of claim8 wherein said chimeric protein comprises an amino acid sequence asgiven in SEQ ID NO:17.
 10. An isolated mutant MHC Class I chimericprotein, wherein said protein comprises a portion mediating binding tothe surfaces of the recombinant yeast cells and a portion whichcomprises a peptide binding region of a MHC Class I protein and whereinsaid chimeric protein is improved in stability as compared with an MHCClass I chimeric protein which is not a mutant chimeric protein.
 11. Theisolated mutant MHC Class I chimeric protein of claim 10 wherein thechimeric protein further comprises a portion comprising an amino acidsequence of a peptide which binds to the peptide binding region of theMHC Class I protein.
 12. The isolated mutant MHC Class I chimericprotein of claim 10 wherein a peptide which binds to the peptide bindingregion of the MHC Class I protein is associated with a neoplastic orinfectious disease.
 13. The isolated mutant MHC Class I chimeric proteinof claim 10 wherein the peptide binding region specifically binds apeptide having the amino acid sequence given in SEQ ID NO:19, SEQ IDNO:22 or SEQ ID NO:24.
 14. The isolated mutant MHC Class I chimericprotein of claim 11 wherein said chimeric protein further comprises adetectable label.
 15. The isolated mutant MHC Class I chimeric proteinof claim 15 wherein the detectable label is a fluorescent moiety, achromophore, a radionuclide, a chemiluminescent agent, a magneticparticle, an enzyme, a cofactor, a substrate or a toxin.
 16. A methodfor detection of a lymphocyte having a T cell receptor protein in abiological sample, said method comprising the steps of contacting thesample with an isolated mutant chimeric protein of claim 14, whereinsaid chimeric protein is complexed to the peptide or wherein thechimeric protein and peptide are covalently bound, wherein said chimericprotein comprises a binding region which specifically binds said T cellreceptor protein under conditions which allow the binding of the T cellreceptor protein to the chimeric protein, and detecting the chimericprotein bound to the T cell receptor protein.
 17. The method of claim 16wherein the T lymphocyte is specific for a neoplastic cell, a tumorcell, a virus-infected cell, a protozoan-infected cell, abacterium-infected cell or a fungus-infected cell.
 18. The method ofclaim 16 or 17 wherein the biological sample is cells, a tissue sample,biopsy material or bodily fluids.
 19. A method for activating orenhancing an immune response to an abnormal cell selected from the groupconsisting of a neoplastic cell, a tumor cell, a virus-infected cell, aparasite-infected cell, a fungus infected cell or a protozoan infectedcell in a human or animal, said method comprising the step ofadministering to the patient a therapeutically effective amount of anisolated mutant MHC Class I chimeric protein or a mutant MHC Class Ichimeric protein/peptide complex which is improved in conformationalstability or improved in binding to T lymphocyte as compared with theMHC Class I chimeric protein which is not mutant, whereby the immuneresponse in the human or animal is activated or enhanced.
 20. The methodof claim 19 wherein the administering is by intravenous, intramuscular,intradermal, subcutaneous or intraperitoneal administration.
 21. Themethod of claim 20 wherein said isolated mutant protein has a portioncomprising an amino acid sequence as given in SEQ ID NO:17.
 22. Themethod of claim 21 wherein said mutant protein binds a peptidecomprising an amino acid sequence as given in SEQ ID NO:19, SEQ ID NO:22or SEQ ID NO:24.
 23. A combinatorial library of peptides anchored to thesurface of yeast cells, each cell displaying a peptide of a uniquesequence anchored to its surface, wherein the peptide is anchored to thesurface of the yeast cell by a mating factor sequence.
 24. Thecombinatorial library of claim 23 wherein the mating factor sequence isan AGA2 sequence.
 25. A method for identifying a peptide which bindsspecifically to an MHC protein comprising the step of contacting adetectable MHC protein with the combinatorial library of claim 24 or 25under conditions which allow binding of the protein and the peptide, anddetecting peptide bound to the MHC protein.